Selective anticancer chimeric peptides which bind neuropilin receptor

ABSTRACT

It is an object of the present invention to provide a substance usable as an anticancer agent or DDS, which has intracellular stability, which is capable of evading side effects from functional disorder with respect to normal cells, or which has instantaneous effect. The inventors developed a novel chimeric peptide targeting cancer cells which overexpress EGFR or the like using a binding peptide such as a peptide sequence binding to EGFR, and a lytic peptide sequence, thereby solving such an object. Particularly, by using a chimeric peptide including an EGF receptor-binding peptide or the like and a cytotoxic peptide, this object was solved.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional patent application of U.S. applicationSer. No. 13/132,580, filed Aug. 15, 2011, now allowed-issued as U.S.Pat. No. 8,436,137 on May 7, 2013; which is a U.S. national stageapplication filed under 35 U.S.C. §371 of International PatentApplication No. PCT/IB2009/055476 accorded an international filing dateof Dec. 3, 2009, which application claims priority from Japanese PatentApplication No. 2009-138729 filed on Jun. 9, 2009, now abandoned, andJapanese Patent Application No. 2008-309176 filed on Dec. 3, 2008, nowabandoned; which applications are incorporated herein by reference intheir entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 920118_(—)401D2_SEQUENCE_LISTING.txt. The textfile is 32 KB, was created on Apr. 4, 2013, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present invention relates to peptidetoxin, a novel targetedtherapeutics.

BACKGROUND ART

Immunotoxins in which monoclonal antibodies or ligands againstoverexpressed proteins on the surface of cancer cells are conjugated toplant or bacterial toxins, have been extensively investigated for theirpossible use as anticancer agents (Non-Patent Document 1). A number ofimmunotoxins have been tested in preclinical and clinical trials, andinterleukin-2-diphteria toxin (IL2-DT; Ontak™) has been approved for thetreatment of chronic T cell lymphocytic leukemia (CLL) (Non-PatentDocument 2, Non-Patent Document 3). In addition, Pseudomonasexotoxin-based immunotoxins including interleukin-4-Pseudomonas exotoxin[IL4 (38-37)-PE38 KDEL] and interleukin-13-Pseudomonas exotoxin(IL13-PE38QQR) have been tested in clinical trials (Non-Patent Document4, Non-Patent Document 5). Both Diphtheria toxin and Pseudomonasexotoxin act by catalytically inactivating the elongation factor 2protein in the ribosome complex, after uptake into lysosomes, activationand translocation into the cytosol. This mechanism of action allows theimmunotoxins to efficiently destroy dormant, non-replicating tumorcells.

Although the targeting approach towards cancer utilizing bacterialtoxin-based immunotoxin is fascinating, its limitation of use lies inthe liver toxicity due to the bacterial toxin and immunogencity causedby the toxin proteins (Non-Patent Document 2, Non-Patent Document 4,Non-Patent Document 6). In addition, molecular size of immunotoxins isgenerally larger compared to chemical compounds or fragment antibodydrugs, which might prevent drugs from efficiently penetrating into tumormass in the human body. To overcome these issues, new generationimmunotoxins with evolutional approach are critically needed.

Epidermal growth factor receptor (EGFR) has been an importanttumor-specific target for many years (Non-Patent Document 7, Non-PatentDocument 8). EGFR plays important roles in cellular growth,differentiation, and migration. Its positive signaling was found tocause increased proliferation, decreased apoptosis, and enhanced tumorcell motility and angiogenesis (Non-Patent Document 9). EGFRoverexpression has been frequently found in a wide spectrum of humantumors of epithelial origin, including breast, lung, gastric,colorectal, prostate, pancreatic and ovarian cancers (Non-PatentDocument 10). All these findings have shown that EGFR is important as atarget for receptor-mediated delivery system of drugs. Recently, severalstudies have reported the successful identification of peptide ligandsof EGFR by screening phage display libraries, implicating possible drugdelivery targeting EGFR (Non-Patent Document 11, Non-Patent Document12).

Therapeutic peptides are increasingly gaining popularity in use in avariety of applications (for example, tumor vaccine (Non-Patent Document13), antimicrobial therapy (Non-Patent Document 14), and nucleic aciddelivery (Non-Patent Document 15)) (Non-Patent Document 16). Inaddition, research and development of new cancer therapy involvingpeptide-based drug has been undertaken (Non-Patent Document 17,Non-Patent Document 18). It is also known that peptide therapeutics arerelatively easily generated using either recombinant or solid-phasechemical synthesis techniques and are generally less expensive whencompared to antibody-based therapeutics. In recent years, it has beenreported that a new lytic-type peptide composed of a 15-amino aciddiastereomeric sequence containing D- and L-forms of leucine and lysinecan disrupt the plasma membrane (Non-Patent Document 19). This peptidekills tumor cells better than normal cells, and disintegrates the cellmembrane in a detergent-like manner. Cell selectivity is probablydetermined predominantly by an increase in the level of acidiccomponents or phosphatidylserine on the cancer cell wall (Non-PatentDocument 19). The diastereomeric sequence preserves activity in serumand in the presence of proteolytic enzymes (Non-Patent Document 20). Ithas been suggested that the peptide's selectivity is probably influencedpredominantly by an increase in the level of acidic components orphosphatidylserine on the cancer cell wall (Non-Patent Document 19).This lytic peptide has selective cytotoxicity between normal and cancercells, but still kills normal cells at a lower concentration, and thusis not suitable for the combination with peptides with targeting moiety.Furthermore, the peptides disclosed in Documents 17, 19 and 20 are notsuitable for molecular targeting, since enhancement of a cell-killingeffect by EGFR targeting is not observed.

Several potential molecular-targeted anticanter marketed drugs inhibitreceptor tyrosine kinase and tumor growth. In some cases, mutations ofkinase-related signal molecule genes in cancer cells result in theresistance to tyrosine kinase inhibitor (TKI) drugs. Recently, it wasrevealed that k-ras mutations are significantly associated with a lackof response to epidermal growth factor receptor (EGFR) TKIs andcetuximab in patients with non-small-cell lung cancer and advancedcolorectal cancer (Non-Patent Document 21). To overcome this criticalissue, development of a novel molecular-targeted anticancer drugdirectly killing cancer cells, which is superior to a signal pathwayblocker is desired.

PRIOR ART DOCUMENTS Non-Patent Documents

-   Non-Patent Document 1: Pastan I. Targeted therapy of cancer with    recombinant immunotoxins. Biochim Biophys Acta 1997; 1333:C1-6-   Non-Patent Document 2: Kawakami K, Nakajima O, Morishita R, Nagai R.    Targeted anticancer immunotoxins and cytotoxic agents with direct    killing moieties. The Sci World J 2006; 6:781-90-   Non-Patent Document 3: Kreitman R J. Immunotoxins for targeted    cancer therapy. AAPS J 2006; 8:E532-51-   Non-Patent Document 4: Rand R W, Kreitman R J, Patronas N,    Varricchio F, Pastan I, Puri R K. Intratumoral administration of    recombinant circularly permuted interleukin-4-Pseudomonas exotoxin    in patients with high-grade glioma. Clin Cancer Res 2000; 6:2157-65-   Non-Patent Document 5:Kunwar S, Prados M D, Chang S M, et al.,    Cintredekin Besudotox Intraparenchymal Study Group. Direct    intracerebral delivery of cintredekin besudotox (IL13-PE38QQR) in    recurrent malignant glioma: a report by the Cintredekin Besudotox    Intraparenchymal Study Group. J Clin Oncol 2007; 25:837-44-   Non-Patent Document 6: Frankel A E, Kreitman R J, Sausville E A.    Targeted toxins. Clin Cancer Res 2000; 6:326-34-   Non-Patent Document 7: Grunwald V, Hidalgo M. Developing inhibitors    of the epidermal growth factor receptor for cancer treatment. J Natl    Cancer Inst 2003; 95:851-67-   Non-Patent Document 8: Janne P A, Engelman J A, Johnson B E.    Epidermal growth factor receptor mutations in non-small-cell lung    cancer: implications for treatment and tumor biology. Clin Oncol    2005; 23:3227-34-   Non-Patent Document 9: Woodburn J R. The epidermal growth factor    receptor and its inhibition in cancer therapy. Pharmacol Ther 1999;    82:241-50-   Non-Patent Document 10: Salomon D S, Brandt R, Ciardiello F,    Normanno N. Epidermal growth factor-related peptides and their    receptors in human malignancies. Crit. Rev Oncol Hematol 1995;    19:183-232-   Non-Patent Document 11: Li Z, Zhao R, Wu X, et al., Identification    and characterization of a novel peptide ligand of epidermal growth    factor receptor for targeted delivery of therapeutics. FASEB J 2005;    19:1978-85-   Non-Patent Document 12: Yao G, Chen W, Luo H, et al., Identification    of core functional region of murine IL-4 using peptide phage display    and molecular modeling. Int Immunol 2005; 18:19-29-   Non-Patent Document 13: Fuessel S, Meye A, Schmitz M, et al.,    Vaccination of hormone-refractory prostate cancer patients with    peptide cocktail-loaded dendritic cells: results of a phase I    clinical trial. Prostate 2006; 66:811-21-   Non-Patent Document 14: Chromek M, Slamova Z, Bergman P, et al., The    antimicrobial peptide cathelicidin protects the urinary tract    against invasive bacterial infection. Nat Med 2006; 12:636-41-   Non-Patent Document 15: Kumar P, Wu H, McBride J L, et al.,    Transvascular delivery of small interfering RNA to the central    nervous system. Nature 2007; 448:39-43-   Non-Patent Document 16: Lien S, Lowman H B. Therapeutic peptides.    Trends Biotechnol 2003; 21:556-62-   Non-Patent Document 17: Ellerby H M, Arap W, Ellerby L M, et al.,    Anti-cancer activity of targeted pro-apoptotic peptides. Nat Med    1999; 5:1032-8-   Non-Patent Document 18: Plescia J, Salz W, Xia F, et al., Rational    design of shepherdin, a novel anticancer agent. Cancer Cell 2005;    7:457-68-   Non-Patent Document 19: Papo N, Shai Y. New lytic peptides based on    the D, L-amphipathic helix motif preferentially kill tumor cells    compared to normal cells. Biochemistry 2003; 42:9346-54-   Non-Patent Document 20: Papo N, Braunstein A, Eshhar Z, Shai Y.    Suppression of human prostate tumor growth in mice by a cytolytic    D-, L-amino acid peptide: membrane lysis, increased necrosis, and    inhibition of prostate-specific antigen secretion. Cancer Res 2004;    64:5779-86-   Non-Patent Document 21: Karapetis, C. S. et al. K-ras mutations and    benefit from cetuximab in advanced colorectal cancer. N Engl J. Med.    359, 1757-1765 (2008)

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

It is an object of the present invention to provide a novelpharmaceutical (for example, anticancer agent) having a new structure.

Means for Solving the Problems

Using the inventors' recent identification information of peptidesequences binding to an EGFR and lytic-type peptide sequence, theinventors have developed a new chimeric peptide targetingEGFR-overexpressed cancer cells, and have found that a chimeric peptideincluding a receptor-binding peptide and a cytotoxic peptide can be usedas a pharmaceutical such as an anticancer agent. This chimeric peptide,termed herein EGFR-targeted peptidetoxin, is preferably composed of areceptor-binding peptide (for example, EGFR-binding moiety) and acytotoxic peptide (for example, cell membrane-lytic moiety) with aspacer (for example, three-glycine spacer). This chimeric peptide ischaracterized in that it is stable when combined with a targeted peptideand has less toxic action on a normal cell line in comparison with theoriginal lytic-type peptide. In the present invention, the inventorsdemonstrated in vitro cytotoxic activity and selectivity of cell deathinduced by a chimeric peptide of the present invention (for example,EGFR-targeted peptidetoxin) in seven human cancer cell lines derivedfrom breast cancer, pancreatic cancer, lung cancer, prostate cancer, andbrain tumor. The inventors have also investigated the interaction ofEGFR-targeted peptidetoxin with cancer cell surface and the mode ofaction of peptidetoxin-induced cancer cell death. In vivo experimentshave also revealed that the chimeric peptide of the present inventionexhibited significant antitumor activity. This has allowed enabled thepeptide to be applied to fields to which it can be applied other thanapplication as an actual pharmaceutical of anticancer agent, forexample, application to screening of a drug or the like, which has notbeen known so far. This is because an application in the form ofscreening for finding a peptide sequence binding to a particular proteinsuch as a receptor, in which a number of types of chimeric peptides ofcandidate sequences and Lytic peptide are synthesized and in which invitro cell-killing effect is an indicator is possible. For example, itis possible to realize a method of screening a pharmaceutical/anticanceragent using an amino acid sequence which targets both EGFR in a cancercell with high EGFR expression and a cancer cell membrane of the cancercell. Lytic-type peptides of conventional arts have selectivecytotoxicity between normal cells and cancer cells, as described above,whereas the peptide of the present invention has reduced toxicity fornormal cells. Thus, the peptide of the present invention has beenrevealed to be suitable for the combination with a peptide and atargeting moiety.

This peptidetoxin is a chemically synthesized peptide composed of atarget-binding peptide and a cytotoxic lytic peptide moiety. As oneexample, an epidermal growth factor receptor (EGFR)-binding peptide wasconjugated to a lytic-type peptide including a cation-rich amino acid,which disintegrates a cell membrane by positive charge to kill a cancercell. In an EGFR-overexpressing cancer cell line, the EGFR-targetedpeptidetoxin induced improvement of IC₅₀ (peptide concentration whichinduces 50% inhibition of proliferation of a control cell) of aboutthree times in comparison with a lytic peptide alone. On the contrary, anormal cell line had low sensitivity to the EGFR-targeted peptidetoxinor lytic peptide alone. In the normal cells, IC₅₀ of four to eight timeshigher than in the cancer cells was exhibited. Interestingly, expressionof EGFR on the cell surface was sufficiently correlated to a degree ofenhancement of the cell-killing effect of the peptidetoxin due to EGFRtargeting, which suggested specificity. Surprisingly, exposure of theEGFR-targeted peptidetoxin for less than 10 minutes was sufficient forkilling over 50% of the cancer cells. Furthermore, the inventors havefound that in cancer cells provided with a peptidetoxin, activation ofpolycaspase and Annexin V-positive expression are induced and thatinduction of an apoptotic mechanism is suggested. In conclusion, apeptidetoxin targeted for a protein highly expressing in a cancer cellmay be a landmark tool for novel targeted therapy of cancer.

Thus, the present invention provides the following.

In a first aspect of the present invention, the present inventionprovides a chimeric peptide including a receptor-binding peptide and acytotoxic peptide.

In one embodiment, the receptor-binding peptide may be EGFreceptor-binding peptide, interleukin-4 (IL-4) receptor-binding peptide,interleukin-13 (IL-13) receptor-binding peptide, neuropilinreceptor-binding peptide, human epidermal growth factor receptor type 2(HER2)-binding peptide, vascular epithelial growth factor receptor(VEGFR)-binding peptide, Transferrin Receptor (TfR)-binding peptide,ephrin B1 (EphB1)-binding peptide, ephrin B2 (EphB2)-binding peptide, aglucose-regulated protein 78 (GRP78)-binding peptide, prostate-specificmembrane antigen (PSMA)-binding peptide or the like.

Discussing from another viewpoint, in the main aspect of the presentinvention, the present invention provides a peptidetoxin, a chimericpeptide including an anticancer targeting peptide and a cellmembrane-lytic peptide moiety.

Specific embodiments in the main aspect of the invention include, forexample, peptidetoxins using a peptide which binds a receptor such asepidermal growth factor (EGF), human epidermal growth factor receptortype 2 (HER), vascular epithelial growth factor receptor 1 (VEGFR1),Transferrin Receptor (TfR), interleukin-4 (IL4), interleukin-13 (IL13),neuropilin (NRP), neuropilin 1 (NRP1)/vascular endothelial growth factorreceptor 2 (VEGFR2), ephrin B1 (EphB1), ephrin B2 (EphB2),glucose-regulated protein (GRP78), prostate-specific membrane antigen(PSMA), or the like. The following are exemplified as chimeric peptides(as used herein, each alphabet indicates one-letter amino acidrepresentation).

EB-Lytic: (SEQ ID NO: 2) YHWYGYTPQNVIGGGKLLLKLLKKLLKLLKKK EB(H2R)-Lytic:(SEQ ID NO: 14) YRWYGYTPQNVIGGGKLLLKLLKKLLKLLKKK HER2-Lytic:(SEQ ID NO: 15) YCDGFYACYMDVGGGKLLLKLLKKLLKLLKKK VEGFR1-Lytic:(SEQ ID NO: 16) WHSDMEWWYLLGGGGKLLLKLLKKLLKLLKKK TfR-Lytic:(SEQ ID NO: 17) THRPPMWSPVWPGGGKLLLKLLKKLLKLLKKK LyticL peptide:(SEQ ID NO: 27) KLLLKLLKKLLKLLKKK IL4-LyticL: (SEQ ID NO: 18)KQLIRFLKRLDRNGGGKLLLKLLKKLLKLLKKK IL13-LyticL: (SEQ ID NO: 19)KDLLLHLKKLFREGQFNGGGKLLLKLLKKLLKLLKKKSema3A-LyticL <binding to human neuropilin-1>: (SEQ ID NO: 20)NYQWVPYQGRVPYPRGGGKLLLKLLKKLLKLLKKK EGFbuf: (SEQ ID NO: 21)YHWYGYTPQNVIGGGGGRLLRRLLRRLLRK

In one embodiment, a cell membrane-lytic peptide moiety used in thepresent invention is composed of an amino acid having a plus charge [K,R or (H)] and a hydrophobic amino acid [L, A, F or the like] and has a10- to 30-amino acids sequence with an amphipathic helix structure.

In one embodiment, an anticancer targeting peptide used in the presentinvention has a binding sequence specific for a receptor with highexpression in cancer cells, and the lytic peptide moiety has a cancercell membrane-lytic sequence and has a spacer.

In one embodiment, a lytic peptide moiety used in the present inventionis composed of an amino acid having a plus charge [K, R or (H)] and ahydrophobic amino acid [L, A, F or the like] and has a 10- to 30-aminoacids sequence with an amphipathic helix structure.

In one embodiment, a spacer used in the present invention is a 0- to5-amino acids sequence consisting of G and P.

In one aspect, the present invention provides a chimeric peptideincluding an EGF receptor-binding peptide and a cytotoxic peptide or acytolytic peptide.

In one embodiment, an EGF receptor-binding peptide used in the presentinvention has the amino acid sequence YHWYGYTPQNVI (SEQ ID NO: 7)wherein each alphabet indicates one-letter amino acid representation, ora modified sequence thereof.

In one embodiment, an EGF receptor-binding peptide used in the presentinvention has the amino acid sequence X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂ (SEQID NO: 8), wherein:

X₁ is Y or an amino acid similar thereto;

X₂ is H or an amino acid similar thereto;

X₃ is W or an amino acid similar thereto;

X₄ is Y or an amino acid similar thereto;

X₅ is G or an amino acid similar thereto;

X₆ is Y or an amino acid similar thereto;

X₇ is T or an amino acid similar thereto;

X₈ is P or an amino acid similar thereto;

X₉ is Q or an amino acid similar thereto;

X₁₀ is N or an amino acid similar thereto;

X₁₁ is V or an amino acid similar thereto; and

X₁₂ is I or an amino acid similar thereto.

In a preferred embodiment, X₁ is Y, or an amino acid similar theretohaving an OH group or an aromatic group;

X₂ is H, or an amino acid similar thereto having a plus charge;

X₃ is W, or an amino acid similar thereto having an aromatic group;

X₄ is Y, or an amino acid similar thereto having an OH group;

X₅ is G, or an amino acid similar thereto having an aliphatic sidechain;

X₆ is Y, or an amino acid similar thereto having an OH group;

X₇ is T, or an amino acid similar thereto having an OH group;

X₈ is P or an imino acid-type amino acid similar thereto;

X₉ is Q or an amide-type amino acid similar thereto;

X₁₀ is N, or an amino acid similar thereto having an OH group;

X₁₁ is V, or an amino acid similar thereto having an aliphatic sidechain; and

X₁₂ is I, or an amino acid similar thereto having an aliphatic sidechain.

In a more preferable example, X₁ is Y, or an amino acid similar theretowhich is S, H or F;

X₂ is H, or an amino acid similar thereto which is R or K;

X₃ is W, or an amino acid similar thereto which is Y, F or H;

X₄ is Y, or an amino acid similar thereto which is S, H or F;

X₅ is G, or an amino acid similar thereto which is A, V, I or L;

X₆ is Y, or an amino acid similar thereto which is S, H or F;

X₇ is T, or an amino acid similar thereto which is S, H or F;

X₈ is P, or an amino acid similar thereto which is hydroxyl proline;

X₉ is Q, or an amino acid similar thereto which is N;

X₁₀ is N, or an amino acid similar thereto which is S, H or F;

X₁₁ is V, or an amino acid similar thereto which is G, A, L or I; and

X₁₂ is I, or an amino acid similar thereto which is G, A, V or L.

In a more preferable embodiment, X₂ is H, or an amino acid similarthereto which is R or K.

In a more preferable embodiment, a chimeric peptide of the presentinvention has the sequence YRWYGYTPQNVI (SEQ ID NO: 9) or YKWYGYTPQNVI(SEQ ID NO: 10).

In one embodiment, a cytotoxic peptide used in the present invention isselected from the group consisting of: cell membrane-lytic peptide, cellmembrane potential-destabilizing peptide, cell membrane-lytic peptideand mitochondrial membrane-disintegrating peptide.

In a preferred embodiment, a cell membrane-lytic peptide used in thepresent invention has a 10- to 20-amino acids sequence consisting onlyof K and L, and the amino acids are L-, D- or D,L-mix amino acids.

In a preferred embodiment, a cell membrane-lytic peptide used in thepresent invention is KLLLKLLKKLLKLLKKK (SEQ ID NO: 48; specifically SEQID NO:1 or 27, or the like), and the amino acids are L-, D- or D,L-mixamino acids.

In a preferred embodiment, a cell membrane potential-destabilizingpeptide used in the present invention is FLKLLKKLAAKLF (SEQ ID NO: 11).

In a preferred embodiment, a cell membrane-lytic peptide used in thepresent invention is RLLRRLLRRLLRRLLRRLLR (SEQ ID NO: 12) orRLLRRLLRRLLRK (SEQ ID NO: 13).

In a preferred embodiment, a mitochondrial membrane-disintegratingpeptide used in the present invention is KLAKLAKKLAKLAK (SEQ ID NO: 4).

In one embodiment, a cytotoxic peptide used in the present invention iscomposed of an amino acid having a plus charge [K, R or (H)] and ahydrophobic amino acid [L, A, F or the like] and has a 10- to 30-aminoacids sequence with an amphipathic helix structure.

In a preferred embodiment, an amino acid having a plus charge used inthe present invention is K, R or H.

In a preferred embodiment, a hydrophobic amino acid used in the presentinvention is I, L, V, A or F.

In a more preferable embodiment, a hydrophobic amino acid used in thepresent invention is KLLLKLLKKLLKLLKKK (SEQ ID NO: 1), wherein eachamino acid is L- or D-amino acid and the underlined letters representD-amino acids.

In one embodiment, a cytotoxic peptide used in the present invention iscomposed of an amino acid having a plus charge [K, R or (H)] and ahydrophobic amino acid [L, A, F or the like] and has a 10- to 30-aminoacids sequence with an amphipathic helix structure.

In one embodiment, an amino acid having a plus charge used in thepresent invention is K, R or H.

In one embodiment, a hydrophobic amino acid used in the presentinvention is I, L, V, A or F.

In one embodiment, a hydrophobic amino acid used in the presentinvention is KLLLKLLKKLLKLLKKK (SEQ ID NO: 1), wherein each amino acidis L- or D-amino acid and the underlined letters represent D-aminoacids.

In one embodiment, a chimeric peptide of the present invention furtherhas a spacer peptide.

In one embodiment, a spacer peptide used in the present inventionexhibits a sequence in which 0 to 4 glycine alone, proline alone orglycine and proline mixed are linked, and is preferably GGG.

In one embodiment, a chimeric peptide of the present invention has thesequence of YHWYGYTPQNVIGGGKLLLKLLKKLLKLLKKK (SEQ ID NO: 2).

In one embodiment, a chimeric peptide of the present invention has thesequence of YRWYGYTPQNVIGGGKLLLKLLKKLLKLLKKK (SEQ ID NO: 43), whereinthe underlined letters represent D-amino acids.

In one embodiment, a receptor-binding peptide of a chimeric peptide ofthe present invention is an interleukin-4 (IL-4) receptor-bindingpeptide and has the amino acid sequence KQLIRFLKRLDRN (SEQ ID NO: 26) ora modified sequence thereof.

In one embodiment, a receptor-binding peptide of a chimeric peptide ofthe present invention is an interleukin-13 (IL-13) receptor-bindingpeptide and has the amino acid sequence KDLLLHLKKLFREGQFN (SEQ ID NO:28) or a modified sequence thereof.

In one embodiment, a receptor-binding peptide of a chimeric peptide ofthe present invention is a neuropilin receptor-binding peptide and hasthe amino acid sequence NYQWVPYQGRVPYPR (SEQ ID NO: 29) or a modifiedsequence thereof.

In one embodiment, a receptor-binding peptide of a chimeric peptide ofthe present invention is a human epidermal growth factor receptor type 2(HER2)-binding peptide and has the amino acid sequence YCDGFYACYMDV (SEQID NO: 30), LLGPYELWELSH (SEQ ID NO: 52), ALVRYKDPLFVWGFL (SEQ ID NO:53), KCCYSL (SEQ ID NO: 54), WTGWCLNPEESTWGFCTGSF (SEQ ID NO: 55),DTDMCWWWSREFGWECAGAG (SEQ ID NO: 56) or a modified sequence thereof.

In one embodiment, a receptor-binding peptide of a chimeric peptide ofthe present invention is a vascular epithelial growth factor receptor 1(VEGFR1)-binding peptide and has the amino acid sequence WHSDMEWWYLLG(SEQ ID NO: 31), VEPNCDIHVMWEWECFERL-NH2 (SEQ ID NO: 32) orGGNECDAIRMWEWECFERL (SEQ ID NO: 33), or a modified sequence thereof.

In one embodiment, a receptor-binding peptide of a chimeric peptide ofthe present invention is a Transferrin Receptor (TfR)-binding peptideand has the amino acid sequence THRPPMWSPVWP (SEQ ID NO: 34) or amodified sequence thereof.

In one embodiment, a receptor-binding peptide of a chimeric peptide ofthe present invention is: a fibroblast growth factor receptor(FGFR)-binding peptide and is MQLPLAT (SEQ ID NO: 5) or AAVALLPAVLLALLAP(SEQ ID NO: 6); neuropilin 1 (NRP1)/vascular endothelial growth factorreceptor 2 (VEGFR2)-binding peptide and is ATWLPPR (SEQ ID NO: 36);ephrin B1 (EphB1)-binding peptide and is EWLS (SEQ ID NO: 37); ephrin B2(EphB2)-binding peptide and is SNEW (SEQ ID NO: 38); interleukin-11receptor (IL11R)-binding peptide and is CGRRAGGSC (cyclic) (SEQ ID NO:22); a glucose-regulated protein 78 (GRP78)-binding peptide and isWDLAWMFRLPVG (SEQ ID NO 39) or CTVALPGGYVRVC (cyclic) (SEQ ID NO: 40); aprostate-specific membrane antigen (PSMA)-binding peptide and isCQKHHNYLC (SEQ ID NO: 35); or a modified sequence thereof.

In one embodiment, a chimeric peptide of the present invention has asequence selected from the group consisting of: SEQ ID NOS: 2, 14, 21,42 and 43, or a modified sequence thereof.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 15 or a modified sequence thereof.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 16 or a modified sequence thereof.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 17 or a modified sequence thereof.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 18 or 44 or a modified sequencethereof.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 19 or a modified sequence thereof.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 20, 46 or 47 or a modified sequencethereof.

In one aspect, the present invention provides a nucleic acid encodingthe chimeric peptide of the present invention.

In another aspect, the present invention provides a vector including anucleic acid which encodes the chimeric peptide of the presentinvention.

In another aspect, the present invention relates to a cell including anucleic acid which encodes the chimeric peptide of the presentinvention.

In another aspect, the present invention relates to a pharmaceutical,preferably a pharmaceutical composition, including the chimeric peptideof the present invention.

In another aspect, the present invention relates to an anticancer agentincluding the chimeric peptide of the present invention.

In another aspect, the present invention relates to a use of thechimeric peptide of the present invention for the manufacture of apharmaceutical composition.

In another aspect, the present invention relates to a use of thechimeric peptide of the present invention for the manufacture of ananticancer agent.

In another aspect, the present invention relates to a method oftreatment including the step of administering the chimeric peptide ofthe present invention.

In another aspect, the present invention relates to a method of treatingcancer including the step of administering the chimeric peptide of thepresent invention.

In one aspect, the present invention relates to a method of screening apharmaceutical using an amino acid sequence targeted by an EGFreceptor-binding peptide of the present invention.

In one aspect, the present invention relates to a method of screening ananticancer agent using an amino acid sequence targeted by an EGFreceptor-binding peptide of the present invention.

Production of various types of chimeric peptides have been attempted. Incomparison with the closest prior art (Ellerby H M, Arap W, Ellerby L M,et al., Nat Med 1999; 5:1032-8; Papo N, Shai Y. Biochemistry 2003;42:9346-54; and Papo N, Braunstein A, Eshhar Z, Shai Y. Cancer Res 2004;64:5779-86), the present invention can be recognized to attain asignificant effect in that it achieved cancer cell targeting andenhancement and immediateness of cell-killing effect with respect tocancer cells as compared to a cancer cell membrane-lytic peptide alone,as a result of chimerization of the present invention. Specifically,although another closest prior art, the targeting approach towardscancer utilizing bacterial toxin-based immunotoxin, is fascinating, itslimitation of use lies in the liver toxicity due to the bacterial toxinand immunogencity caused by the toxin proteins (Non-Patent Document 2,4, 6). In addition, molecular size of immunotoxins is generally largercompared to chemical compounds or fragment antibody drugs, which mightprevent drugs from efficiently penetrate into tumor mass in the humanbody (Non-Patent Document 2, 4, 6). These documents merely present anissue of protein formulations such as immunotoxin, and present no meansfor solving the issue. To overcome this issue, a new generationimmunotoxins with evolutional approach are critically needed.

In all these aspects, it is understood that each embodiment describedherein may be applied in other aspect as long as it is applicable.

Effect of the Invention

A substance usable as an anticancer agent or DDS which has intracellularstability, capable of evading side effects from functional disorder withrespect to normal cells, or which has instantaneous effects is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows cell-killing effect of EGFR-binding (EB)-lytic chimericpeptide or lytic peptide alone in various human cancer cell lines.Cancer cell line H322, BT-20, H460, U251, BxPC-3, SU.86.86 or LNCaP wascultured with various concentrations (0 to 30 μM) of EB-lytic chimericpeptide or lytic peptide for 0.72 hours, and cytotoxic activity wasassessed using WST-8 reagent. The results are represented as mean oftriplicate measurements±SD (bar). This assay was repeated three times.Black circle, EB-lytic chimeric peptide; white circle, lytic peptide.

FIG. 1B shows a cell-killing effect of EB-lytic chimeric peptide orlytic peptide alone in various human normal cell lines. Normal celllines MRC-5, WI-38 or HEK293 was cultured with various concentrations (0to 100 μM) of the peptides for 72 hours, and cytotoxic activity wasassessed. The results are represented as mean of triplicatemeasurements±SD (bar). This assay was repeated three times. Blackcircle, EB-lytic chimeric peptide; white circle, lytic peptide.

FIG. 1C shows a cell-killing effect of EB-original lytic chimericpeptide or original lytic peptide alone in various human cancer cellsand normal cells. Cancer cell line H322 and normal cell line MRC-5 werecultured with various concentrations (0 to 30 μM) of EB-original lyticchimeric peptide or original lytic peptide alone, and cytotoxicity assaywas performed as described above. Black circle, EB-original lyticchimeric peptide; white circle, original lytic peptide.

FIG. 1D shows designing and secondary structure analysis of a novelchimeric peptide using CD spectrum. The upper shows wheel projection ofSchiffer Edmundson for EGFR-binding (EB) peptide conjugated to a lyticpeptide (a) reported by Papo and Shai (original lytic peptide) or to anewly designed lytic peptide (b). In the peptide sequences, underlineditalic letters represent D-amino acids. Bold letters in the wheeldiagrams represent hydrophilic amino acids (mainly Lys). Arrowsrepresent a direction of a hydrophilic surface of these peptides. CDspectra of EB-original lytic peptide (c) and EB-lytic peptide (d) in thepresence of PC SUV or PC/PS (4:1) SUV are shown. Concentrations ofpeptide and lipid were respectively 50 μM and 4 mM. The amino acidsequence shown in FIG. 1D, part a, is SEQ ID NO:42, and the amino acidsequence shown in FIG. 1D, part b, is SEQ ID NO: 2.

FIG. 1E shows resistance to tyrosine kinase inhibitor (TKI) of k-rasmutated cancer cell line. Wild-type k-ras cancer cell lines (H322 (blackcircle) and BT-20 (black triangle) and mutated k-ras cancer cell lines(MDA-MB-231 (white circle), HCT116 (white triangle), SW837 (whiterhomboid) and DLD-1 (x)) were cultured with various concentrations oferlotinib (left; 0 to 80 μM) or anti-EGFR antibody (right; 0 to 20 μM)for 72 hours, and cytotoxic activity was assessed using WST-8 reagent.The vertical axis shows cell viability (%) with respect to control, andthe horizontal axis shows concentration of erlotinib (left) andanti-EGFR antibody (right). The assay was repeated three times, and theresults are represented as mean of the triplicate measurements±SD (bar).

FIG. 1F shows comparison of cytotoxicity between TKI and EB-lyticchimeric peptide in wild-type k-ras cell line. Cancer cell lines (H322,BT-20 and BxPC-3) and lung normal cell line (MRC-5) were cultured withvarious concentrations of TKI (erlotinib (white circle), gefitinib(white triangle) and PD153035 (white rhomboid); 0 to 20 μM) or EB-lyticchimeric peptide (black circle; 0 to 20 μM) for 72 hours, and cytotoxicactivity was assessed using WST-8 reagent. The vertical axis shows cellviability (%) with respect to control, and the horizontal axis showsconcentration (μM) of TKI and EB-lytic. The assay was repeated threetimes, and the results are represented as mean of the triplicatemeasurements±SD (bar).

FIG. 1G shows that the treatment of EB-lytic chimeric peptide results insufficient cytotoxic activity to TKI-resistant cancer cell lines havingk-ras mutation. Four types of mutated k-ras cancer cell lines(MDA-MB-231) were cultured with various concentrations of TKI (erlotinib(white circle), gefitinib (white triangle) and PD153035 (whiterhomboid); 0 to 20 μM) or EB-lytic chimeric peptide (black circle; 0 to20 μM) for 72 hours, and cytotoxic activity was assessed using WST-8reagent. The vertical axis shows cell viability (%) with respect tocontrol, and the horizontal axis shows concentration (M) of TKI andEB-lytic. The assay was repeated three times, and the results arerepresented as mean of the triplicate measurements±SD (bar).

FIG. 2A shows that enhancement of cell-killing effect by EB-lyticchimeric peptide depends on expression of EGFR on a cell surface.Correlation with relative mean fluorescence intensity of EGFR antibodybinding of IC₅₀ for EB-lytic chimeric peptide (left), IC₅₀ for lyticpeptide (center), or IC₅₀ ratio of lytic peptide with respect toEB-lytic chimeric peptide (right) in seven cancer cell lines and threenormal cell lines.

FIG. 2B shows that cell-killing effect of EB-lytic chimeric peptide toBxPC-3 cells can be inhibited by addition of EGFR antibody orrecombinant EGF protein. Inhibition of cell-killing effect of EB-lyticchimeric peptide to BxPC-3 cells was evaluated by addition of apolyclonal anti-EGFR antibody or recombinant EGF protein one hour priorto exposure to the peptide. *P<0.05, **P<0.01.

FIG. 3A shows binding properties of EB-lytic chimeric peptide and lyticpeptide to EGFR protein. Samples of serially diluted EB-lytic chimericpeptide (27 μM to 13 μM) or lytic peptide (24 μM to 12 μM) were analyzedon sensor surfaces.

FIG. 3B shows interaction profile for binding of lytic peptide alone tocell surface membrane proteins extracted from H322 or MRC-5 cells.Samples of serially diluted membrane proteins (0.1 mg/ml to 0.025 mg/ml)were analyzed on sensor surfaces.

FIG. 3C shows interaction profile for binding of EB-lytic chimericpeptide to cell surface membrane proteins extracted from H322 or MRC-5cells. Samples of serially diluted membrane proteins (0.1 mg/ml to 0.025mg/ml) were analyzed on sensor surfaces.

FIG. 3D shows relative K_(D) values of EB-lytic chimeric peptide (blackcolumns) or lytic peptide alone (white columns) to cell surface membraneproteins extracted from H322, BT-20 or MRC-5 cells.

FIG. 4A shows that EB-lytic chimeric peptide induces rapid killing ofcancer cells. H322 and BT-20 cells were treated with EB-lytic chimeric(black columns) or lytic peptide (white columns) for 10 minutes, 30minutes, one hour or 48 hours, and then the medium containing peptideswas replaced with a fresh medium, and culturing was further performedfor 48 hours. The cells were analyzed for cell viability using WST-8.

FIG. 4B shows permeabilization of cell membranes by EB-lytic chimericpeptide in MDA-MB-231 breast cancer cells. Cells (3×10⁴ cells/ml) incalcein solution for 0 minute, two minutes, five minutes, 10 minutes and20 minutes after addition of EB-lytic peptide-TAMRA at a finalconcentration of 10 μM. Arrows and arrowheads indicate permeated cellsand peptides which permeated a membrane, respectively.

FIG. 4C shows permeabilization of cell membranes by lytic peptide inMDA-MB-231 breast cancer cells. Cells (3×10⁴ cells/ml) in calceinsolution for 0 minute, two minutes, five minutes, 10 minutes and 20minutes after addition of lytic peptide-TAMRA at a final concentrationof 10 μM.

FIG. 4D shows inhibition of cellular growth by EB-lytic chimericpeptide. H322 cells were cultured for 10 days in a medium containingvarious concentrations (0 to 22.5 μM) of EB-lytic chimeric peptide.After staining with crystal violet, colonies composed of at least 50cells were scored, and the results are represented as percentagerelative to untreated cells, based on the number of colonies. Untreatedcells formed 117±10 colonies. Data are means of duplicate measurements;bars show SD.

FIG. 5 shows that EB-lytic chimeric peptide induces caspase activationand Annexin V-positive expression in cancer cells. BT-20 cells incubatedwith EB-lytic chimeric peptide (5 μM) were analyzed after two hours bydual-color flow cytometry for Annexin V labeling (upper panel) orcaspase activity by DEVDase activity (lower panel) in the green channel,and for PI staining in the red channel. The value indicates thepercentage of cells in each quadrant.

FIG. 6 shows in vitro cellular growth inhibition of H322 lung cancercells. H322 cells were cultured in a medium containing variousconcentrations (0 to 22.5 μM) of EB-chimeric peptide or lytic peptidealone for 10 days. After staining with crystal violet, colonies composedof at least 50 cells were scored, and the results are represented aspercentage of the number of colonies (100%, untreated cells) Untreatedcells formed 117±10 colonies. Data are means of duplicate measurements;bars show SD.

FIG. 7A shows the results of binding affinity BIACORE analysis forEGFR-binding sequence mutant peptide and recombinant human EGFR. Mutantswere chemically synthesized by changing the second H having charge inthe wild-type EGFR-binding peptide sequence into K or R, and bindingaffinity with EGFR was assessed using enhancement of biosensor responseof BIACORE as an indicator.

FIG. 7B shows the results of comparison of cell-killing effect betweenR-mutant peptide which had high binding affinity with EGFR and thecancer cell membrane-lytic peptide alone. Human lung cancer cell lineH322 was cultured with serially diluted concentrations (0 to 30 μM) oftwo peptides for 72 hours, and cell-killing effect was assessed usingWST-8 reagent. These results are represented as mean of triplicatemeasurements±SD (bar), and the assay was repeated three times. Triangle,EB-chimeric peptide; black circle: R-mutant EB-chimeric peptide; whitecircle, membrane-lytic peptide.

FIG. 8A shows binding properties of IL4R-binding peptide and scramblesequence peptide to recombinant human IL4R. Samples of serially dilutedIL4R-binding peptide (29 μM to 7.4 μM) were analyzed on sensor surfaces.

FIG. 8B shows the results of comparison of cell-killing effect betweenIL4R-targeted cancer cell membrane-lytic chimeric peptide (IL4-LyticL;quadrangle) and the cancer cell membrane-lytic peptide alone (LyticL;rhomboid). Human breast cancer cell line MDA-MB-231 (left) and humanpancreatic cancer cell line BxPC-3 (right) were cultured with seriallydiluted concentrations (0 to 10 μM) of two peptides for 72 hours, andcell-killing effect was assessed using WST-8 reagent. The assay wasrepeated three times, and the results are represented as mean oftriplicate measurements±SD (bar).

FIG. 8C shows that IL4R-targeted cancer cell membrane-lytic chimericpeptide (IL4-LyticL) induces rapid killing of cancer cells. BxPC-3 cellswere treated with 10 μM of IL4-LyticL (black columns) or LyticL (graycolumns) for two minutes, five minutes, 10 minutes, 30 minutes or onehour, and then the medium containing peptides was replaced with a freshmedium and culturing was further performed for 48 hours. The cells wereanalyzed for cell viability using WST-8.

FIG. 9A shows binding properties of IL13R-binding peptide and scramblesequence peptide to recombinant human IL13R. Samples of serially dilutedIL13R-binding peptide (24 μM to 6.0 μM) were analyzed on sensorsurfaces.

FIG. 9B shows the results of comparison of cell-killing effect betweenIL13R-targeted cancer cell membrane-lytic chimeric peptide (IL13-LyticL)and the cancer cell membrane-lytic peptide alone (LyticL). Human braintumor cell line U251 (left) and human head and neck cancer cell lineHN-12 (right) were cultured with serially diluted concentrations (0 to20 μM) of two peptides for 72 hours, and cell-killing effect wasassessed using WST-8 reagent. The assay was repeated three times, andthe results are represented as mean of triplicate measurements±SD (bar).Rhomboid, LyticL; quadrangle, IL13-LyticL.

FIG. 9C shows that IL13R-targeted cancer cell membrane-lytic chimericpeptide (IL13-LyticL) induces rapid killing of cancer cells. U251 cellswere treated with 10 μM of IL13-LyticL (black columns) or LyticL (graycolumns) for two minutes, five minutes, 10 minutes, 30 minutes, or onehour to 24 hours, and then the medium containing peptides was replacedwith a fresh medium and culturing was further performed for 48 hours.The cells were analyzed for cell viability using WST-8.

FIG. 10A shows binding properties of neuropilin-1 (NRP1)-binding peptideand scramble sequence peptide to recombinant human NRP1. Samples ofserially diluted NRP1-binding peptide (24 μM to 6.0 μM) were analyzed onsensor surfaces.

FIG. 10B shows the results of comparison of cell-killing effect betweenNRP1-targeted cancer cell membrane-lytic chimeric peptide(Sema3A-LyticL; black circle) and the cancer cell membrane-lytic peptidealone (LyticL; white circle). Human pancreatic cancer cell line SU8686(left) and human breast cancer cell line SKBR-3 (right) were culturedwith serially diluted concentrations (0 to 10 μM) of two peptides for 72hours, and a cell-killing effect was assessed using WST-8 reagent. Theassay was repeated three times, and the results are represented as meanof triplicate measurements±SD (bar).

FIG. 11A shows the results of comparison of cell-killing effect betweencell membrane-lytic and nucleic acid-binding sequence (buf; whitecircle) and EGFR-targeted chimeric peptide thereof (EGFbuf; blackcircle). Human lung cancer cell line H322 (left) and human prostatecancer cell line DU145 (right) were cultured with serially dilutedconcentrations (0 to 30 μM) of two peptides for 72 hours, andcell-killing effect was assessed using WST-8 reagent. The results arerepresented as mean of triplicate measurements±SD (bar).

FIG. 11B shows the results of comparison of cell-killing effect of theEGFR-targeted chimeric peptide (EGFbuf) to lung cancer cell line H322(white circle) and lung normal cell line MRC-5 (black circle). The twocells were cultured with serially diluted concentrations (0 to 20 μM) ofEGFbuf peptide for 72 hours, and cell-killing effect was assessed usingWST-8 reagent. The results are represented as mean of triplicatemeasurements±SD (bar).

FIGS. 12A and 12B show the results of comparison of cell-killing effectof targeted chimeric peptide by cancer cell membrane-lytitc sequence(D,L-mix; the same sequence as in Example 1) alone and three sequencesbinding to a receptor with high expression in cancer cells (her2, VEGFreceptor, Transferrin receptor). Human lung cancer cell line H322 (FIG.12A) or human lung normal cell line MRC-5 (FIG. 12B) were cultured withserially diluted concentrations (0 to 80 μM) of the four peptides for 72hours, and cell-killing effect was assessed using WST-8 reagent. Theassay was repeated three times, and the results are represented as meanof triplicate measurements±SD (bar). Black triangle: TfR-Lytic; whitecircle: Her2-Lytic; white triangle: VEGFR1-Lytic; black circle: Lytic.

FIG. 13 shows antitumor effect of the same EB-lytic chimeric peptide asin Example 1 in mouse models bearing human pancreatic cancer cell lineBxPC-3. Human pancreatic cancer cell line BxPC-3 was subcutaneouslytransplanted to nude mice. From day 5 after the transplantation, thepeptide was intratumorally administered three times per week for threeweeks. The results are represented as mean of four mice of each group±SD(bar). White circle is a group administered with a solvent, whitetriangle is a group administered with 0.3 mg/kg of the EB-lytic chimericpeptide, and black circle is a group administered with 1 mg/kg of theEB-lytic chimeric peptide.

FIG. 14 shows antitumor effect of systemic administration of the sameEB-lytic chimeric peptide as in Example 1 in mouse models bearing humanpancreatic cancer cell line BxPC-3. Human pancreatic cancer cell lineBxPC-3 was subcutaneously transplanted to nude mice. From day 5 afterthe transplantation, the peptide was intravenously administered threetimes per week for three weeks. The results are represented as mean ofthree mice of each group±SD (bar). White circle is a group administeredwith saline, white triangle is a group administered with 1 mg/kg of theEB-lytic chimeric peptide, and black circle is a group administered with5 mg/kg of the EB-lytic chimeric peptide.

FIG. 15A shows antitumor effect of the same EB-lytic chimeric peptide asin Example 1 in mouse models bearing human pancreatic cancer cell lineBxPC-3 (upper) and mouse models bearing human breast cancer cell lineMDA-MB-231 (lower). BxPC-3 pancreatic cancer cells or breast cancercells were subcutaneously transplanted to athymic nude mice. As shown bythe arrow, from day 5, saline (control (white circle)) or EB-lyticpeptide (2 mg/kg (black quadrangle), 5 mg/kg (white quadrangle) or 10mg/kg (black triangle)) was intravenously administered. Each group isformed by six animals (n=6), and the experiment was repeated two times.The results are represented as mean±SD.

FIG. 15B shows reduction of MDA-MB-231 tumor in the athymic nude miceafter the intravenous treatment. MDA-MB-231 cells were dorsallytransplanted to the mice. After engrafting, the mice were treated byintravenous injection of saline (left panel) or EB-lytic peptide (rightpanel). Arrow indicates location of tumor.

FIG. 15C shows histological test for MDA-MB-231 tumor after thetreatment with the same EB-lytic chimeric peptide as in Example 1.Formalin-fixed and paraffin-embedded tumor sections from the animalstreated with saline (left panel) or EB-lytic chimeric peptide (rightpanel) were stained with hematoxylin, and analyzed with opticalmicroscope.

FIG. 16A shows binding analysis using BIACORE system.

FIG. 16B shows secondary structure analysis for EB-lytic peptide (dottedline) and EB(H2R)-lytic peptide (solid line) using CD spectrum. Thepeptide concentrations were 50 μM.

FIG. 16C shows cytotoxicity of the lytic peptide (rhomboid), EB-lyticpeptide (quadrangle) and EB(H2R)-lytic peptide (triangle) in BT20 cells.

FIG. 17A shows that the newly designed lytic peptide is suitable forchimeric peptides for enhancing cytotoxic activity to cancer cells.Cancer cell lines H322, BT-20, U251, BxPC-3, SU8686 and LNCaP werecultured with various concentrations (0 to 20 μM) of EB-lytic chimericpeptide (black circle) or EB(H2R)-lytic chimeric peptide (white circle),and cytotoxic activity was assessed using WST-8 reagent.

FIG. 17B shows cytotoxicity of EB(H2R)-lytic chimeric peptide orEB-lytic peptide in various human normal cell lines. Normal cell linesMRC-5 and HEK293 were cultured with various concentrations (0 to 20 μM)of the aforementioned peptides, cytotoxic activity was assessed, andcytotoxicity assay was performed as described above. White circle,EB(H2R)-lytic peptide; black circle, EB-lytic peptide.

FIG. 18 shows that the EB-lytic chimeric peptide disintegrates the cellmembrane to induce rapid killing of cancer cells. H322 cells weretreated with 10 μM of EB-lytic chimeric peptide (white columns) orEB(H2R)-lytic chimeric peptide (black columns) for two minutes, fiveminutes, 10 minutes, 30 minutes, one hour, two hours or 24 hours, andthen the medium containing peptides was replaced with a fresh medium.The cells were further cultured for 24 h. The cells were analyzed forcell viability using WST-8. The results are represented as mean±SD(bar).

FIG. 19A shows permeation of cell membrane by Lytic peptide in H322 lungcancer cells. Confocal microscopic images of cells in calcein solutionof 0 minute, two minutes, five minutes, 10 minutes and 20 minutes afteraddition of lytic peptide at final concentration of 10 μM are shown.From the top, images of Lytic peptide alone, EB-Lytic peptide and EB(H2R)-Lytic peptide are shown. These images indicate that the number ofcells which became green (cells with the membrane disintegrated) forLytic peptide alone did not change over time, whereas the number ofcells which became green increased over time for EB-Lytic peptide andEB(H2R)-Lytic peptide and the number increases in shorter time forEB(H2R)-Lytic peptide than for EB-Lytic peptide.

FIG. 19B shows a graph showing the percentage of influx of medium to thecells over time as calculated from the results of FIG. 19A. Also fromthis figure, it is seen that the percentage of influx of medium to thecells was more rapid for EB (H2R)-Lytic peptide (white quadrangle) thanfor EB-Lytic peptide (black quadrangle).

FIG. 20A shows that EB (H2R)-Lytic peptide induces Annexin V-positiveexpression more strongly than EB-Lytic peptide in cancer cells. Humanbreast cancer cell line BT20 cells were incubated with EB-Lytic peptideor EB (H2R)-Lytic peptide (5 μM of each) at 37° C. for two hours, andanalyzed by dual-color flow cytometry for Annexin V labeling.

FIG. 20B shows that EB (H2R)-Lytic peptide induces caspase 3 & 7activity more strongly than EB-Lytic peptide in cancer cells. Humanbreast cancer cell line BT20 cells were incubated with EB-Lytic peptideor EB (H2R)-Lytic peptide (5 μM of each) at 37° C. for two hours, andanalyzed by calboxyfluorescein FLICA polycaspase assay for caspase 3 & 7activity.

FIG. 21 shows antitumor effect of EB-Lytic peptide and EB (H2R)-Lyticchimeric peptide in mouse models bearing human breast cancer cell lineMDA-MB-231. MDA-MB-231 breast cancer cells were subcutaneouslytransplanted to athymic nude mice, and as shown by arrow in the graph,from day 5 after the transplantation, saline (control: black circle),EB-Lytic peptide (1 mg/kg; quadrangle) or EB(H2R)-Lytic chimeric peptide(1 mg/kg; triangle) was intravenously injected. Each group was formed ofsix animals (n=6). The vertical axis shows a volume of tumor (mm³) andthe horizontal axis shows the number of days (day) after thetransplantation of breast cancer cells. The data is represented asmean±SD. It is seen that the chimeric peptide in which the secondposition of EB peptide was changed from H into R (EB(H2R)-Lytic) has invivo antitumor effect higher as compared to EB-Lytic chimeric peptide(EB-Lytic).

FIG. 22A shows that TfR-lytic peptide is suitable for chimeric peptidefor enhancing cytotoxic activity to cancer cells. Cancer cell linesT47D, MDA-MB-231 and SKBR-3 were cultured with various concentrations (0to 30 μM) of TfR-lytic chimeric peptide (white quadrangle) or lyticpeptide (black quadrangle) for 72 hours, and cytotoxic activity wasassessed with WST-8 reagent.

FIG. 22B shows cytotoxicity of TfR-lytic chimeric peptide in variousnormal cell lines. Normal cell lines MRC-5, PE and HC were cultured withvarious concentrations (0 to 100 μM) of the aforementioned peptides for72 hours, and cytotoxic activity was assessed. The absolute valueobtained from untreated cells was defined as 100%. Black quadrangle,TfR-lytic chimeric peptide; white quadrangle, lytic peptide.

FIG. 23 shows correlation between IC₅₀ values related to enhancement ofcytotoxicity by addition of TfR-lytic chimeric peptide. IC₅₀ ofTfR-lytic chimeric peptide (A) and IC₅₀ of lytic peptide (B) or IC₅₀ratio of lytic peptide/TfR-lytic chimeric peptide (C) and meanfluorescence intensity of TfR monoclonal antibody binding for sevencancer cell lines.

FIG. 24A shows that TfR-lytic peptide disintegrates the cell membrane torapidly kill T47D cancer cells. T47D cells were exposed to TfR-lyticchimeric peptide (black columns) or lytic peptides (white columns) forvarious periods of time (1 to 180 minutes), and after exposure for acertain period of time, the medium was replaced with a fresh medium. Thecells were finally cultured for 72 hours, and cell viability wasanalyzed using WST-8. The results are represented as mean±SD (bar).

FIG. 24B shows permeabilization of membrane by TfR-lytic chimericpeptide in T47D breast cancer cells. Cells in calcein solution of 0minute, five minutes, 10 minutes and 15 minutes after addition ofTfR-lytic chimeric peptide at concentration of 10 μM are shown. Arrowshows permeated cells and peptide which permeated the membrane.

FIG. 25A shows inhibition of cell viability of cancer cell lines byTfR-lytic chimeric peptide. Before the treatment with peptide (5 μM),T47D cells were incubated with increasing concentration of anti-TfRmonoclonal antibody (black columns) or nonspecific mouse IgG1 (isotypecontrol; white columns) for three hours.

FIG. 25B shows cytotoxicity of siRNA or scramble sequence (sc) RNA tocancer cell lines. T47D cells and MDA-MB-231 cells were transfected withsiRNA or scRNA. Four days after the transfection, the levels of targetgene in the cells were analyzed by flow cytometry analysis (data notshown), and the percentage of inhibition was assessed using WST-8reagent. The assay was repeated three times, and the results arerepresented as means of triplicate measurements±SD (bar).

FIG. 26A shows that TfR-lytic chimeric peptide induced AnnexinV-positive expression in cancer cells. T47D cells and PE cells wereincubated with TfR-lytic chimeric peptide (10 μM) and lytic peptide (10μM). After two hours, dual-color flow cytometry analysis was performedfor Annexin V-positive in the green channel, and for propidium iodidestaining in the red channel.

FIG. 26B shows that TfR-lytic chimeric peptide induced caspaseactivation in cancer cells. T47D cells and PE cells were incubated withTfR-lytic chimeric peptide (10 μM) and lytic peptide (10 μM). After twohours, dual-color flow cytometry analysis was performed for caspase 3 &7 activity in the green channel, and for propidium iodide staining inthe red channel.

FIG. 26C shows comparison of action of various lytic peptides in cancercell lines. T47D cells labeled with mitochondrial transmembranepotential-dependent fluorochrome JC-1 were untreated (untreated: upperleft panel) or treated with Staurosporine (control mitochondrialmembrane potential: upper right panel), lytic peptide (lower left panel)or TfR-lytic chimeric peptide (lower right panel). After two hours, byflow cytometry, distinctive ratio in changes in red fluorescence orgreen fluorescence indicating transmembrane potential was analyzed.

FIG. 26D shows Western blot analysis for release of cytochrome c incells treated with various lytic peptides. T47D cells were incubatedwith Staurosporine, TfR-lytic chimeric peptide (10 μM) and lytic peptide(10 μM). After two hours, cytoplasm extract was isolated, and by Westernblot analysis using an antibody to cytochrome c, release of cytochrome cwas investigated.

FIG. 27A shows in vivo antitumor activity of TfR-lytic chimeric peptide(intratumoral injection). MDA-MB-231 breast cancer cells weresubcutaneously transplanted to athymic nude mice. As shown by arrow,from day 5, saline (control (black circle)) or TfR-lytic peptide (0.3mg/kg (black rhomboid), 1 mg/kg (white triangle) or 3 mg/kg (whitequadrangle)) was intratumorally injected. Each group was formed by threeanimals (n=3). The data is represented as mean±SD.

FIG. 27B shows in vivo antitumor activity of TfR-lytic chimeric peptide(intravenous injection). MDA-MB-231 cells were subcutaneouslytransplanted to athymic nude mice. As shown by arrow, from day 5, saline(control (black circle)) or TfR-lytic peptide (0.5 mg/kg (whitetriangle), 1 mg/kg (black rhomboid), 2 mg/kg (white quadrangle) or 5mg/kg (white circle)) was intravenously injected. Each group was formedby three animals (n=3). The data is represented as mean±SD.

FIGS. 28A-B shows three-dimensional composite structure of interleukin-4(IL4) and interleukin-4 receptor a helix (IL-4Ra). (FIG. 28A showsentire structure of IL4 and IL-4Ra. FIG. 28B shows an enlarged region ofthe position of interface between IL4 and IL-4Ra. A residue importantfor binding IL4 to IL-4Ra is represented by a black letter.

FIGS. 29A-D shows selective toxicity of the designed chimeric peptideconjugated to L,D-lytic peptide [IL4-Lytic(L,D)] between normal cellsand cancer cells. Normal cell line WI-38 (FIGS. 29A and 29B) and cancercell line KCCT873 (FIGS. 29C and 29D) were cultured with variousconcentrations (0 to 30 μM) of IL4-lytic (L) chimeric peptide, lytic (L)peptide, IL-4-lytic (L,D) chimeric peptide or lytic (L, D) peptide for72 hours. Cytotoxic activity was assessed using WST-8 reagent. Whitequadrangle, lytic (L) peptide; black quadrangle, IL4-lytic (L) peptide;white triangle, lytic (L,D) peptide; black triangle, IL4-lytic (L,D)peptide.

FIG. 30 shows detection of IL-4Rα expression on cell surface of cancercell lines. Total RNA from A172, BxPC-3 and normal cell line HEK293 werereverse-transcribed to cDNA, and then detection was performed byquantitative PCR using IL-4Rα-specific primer.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internalstandard.

FIGS. 31A-B shows that IL4-Lytic (L, D) chimeric peptide rapidly killscancer cells. PE cells (FIG. 31A), KCCT873 cells and BxPC-3 cells (FIG.31B) were treated with IL4-lytic (L, D) chimeric peptide (black columns)or lytic (L,D) peptide (white columns) for two minutes, five minutes, 10minutes, 30 minutes, one hour or two hours. The medium containingpeptides was replaced with a fresh medium, and the cells were furthercultured for 72 hours. Cell viability was determined using WST-8reagent. The results are represented as mean±SD (bar).

FIG. 32 shows that IL4-Lytic (L,D) chimeric peptide induces AnnexinV-positive expression of cancer cells. PE cells which are normal cells(upper) and KCCT873 cells which are cancer cells (lower) were incubatedwith IL4-lytic (L,D) chimeric peptide or lytic (L,D) peptide (10 μM) fortwo hours. Subsequently, dual-color flow cytometry analysis wasperformed for Annexin V labeling in green channel and PI staining in redchannel. Percentage of cells in each quadrant is shown.

FIG. 33A shows in vivo antitumor activity of IL4-Lytic (L,D) chimericpeptide (intratumoral injection). MDA-MB-231 breast cancer cells weresubcutaneously transplanted to athymic nude mice. As shown by arrow,from day 5, saline (NaCl; control (white rhomboid)) or IL4-lytic (L,D)peptide (0.5 mg/kg (black quadrangle) or 2 mg/kg (black triangle)) wasintratumorally injected. Each group was formed by three animals (n=3).

FIG. 33B shows in vivo antitumor activity of IL4-lytic (L,D) chimericpeptide (intravenous injection) MDA-MB-231 breast cancer cells weresubcutaneously transplanted to athymic nude mice. As shown by arrow,from day 5, saline (control (white rhomboid)) or IL4-lytic (L,D) peptide(2 mg/kg (black quadrangle) or 5 mg/kg (black circle)) was intravenouslyinjected. Each group was formed by three animals (n=3). The data isrepresented as mean±SD (bar).

FIG. 34A shows cytotoxic activity of Sema3A-nLytic for various celllines. Pancreas cell line was cultured with various concentrations (0 to50 μM) of Sema3A-nLytic peptide or nLytic peptide alone for 48 hours,and cytotoxic activity was assessed using WST-8 reagent. Rhomboid,nLytic peptide; quadrangle, Sema3A-nLytic (363-377) peptide; triangle,Sema3A-nLytic (371-377) peptide.

FIG. 34B shows cytotoxic activity of Sema3A-nLytic for various celllines. Breast cancer cells were cultured with various concentrations (0to 20 μM) of Sema3A-nLytic (371-377) or Sema3A-nLytic (363-377) for 48hours, and cytotoxic activity was assessed using WST-8 reagent. Theassay was repeated three times, and the results are represented as meansof triplicate measurements±SD (bar). Quadrangle, Sema3A-nLytic (363-377)peptide; triangle, Sema3A-nLytic (371-377) peptide.

FIG. 34C shows cytotoxic activity of Sema3A-nLytic for various celllines. Two cells (left: HuCCT1; right: HEK293) were cultured withvarious concentrations (0 to 20 μM) of Sema3A-nLytic (363-377) orSema3A-nLytic (371-377) for 48 hours, and cytotoxic activity wasassessed using WST-8 reagent. The assay was repeated three times, andthe results are represented as mean of triplicate measurements±SD (bar).Quadrangle, Sema3A-nLytic (363-377) peptide; triangle, Sema3A-nLytic(371-377) peptide.

FIGS. 35A-D shows analysis of interaction with neuropilin-1 (NRP1) ofwild-type and some mutated peptides of Sema3A using BIACORE. (FIG. 35A)shows binding of Sema3A wild-type peptide to NRP1 protein. Samples ofserially diluted various concentrations (1.6 μM, 13 μM, 26 μM) of Sema3Awild-type peptide were analyzed on parallel sensor surface. (FIG. 35B)shows binding of wild-type and mutated peptides of Sema3A (R372K andR372K/R377K) to NRP1 protein. Samples of various serially diluted Sema3Apeptides (26 μM) were analyzed on parallel sensor surface. (FIG. 35C)shows outline of binding ability of various Sema3A peptides to NRP1protein. (FIG. 35D) shows peptide sequence of wild-type and mutatedpeptides of Sema3A. The sequence identifiers corresponding to the aminoacid sequences shown in FIG. 35D are as follows: Sema3A wild peptide,SEQ ID NO:29; R372K, SEQ ID NO:60; V373A, SEQ ID NO:61; P374T, SEQ IDNO:62; Y375L, SEQ ID NO:63; P376T, SEQ ID NO:64; R377K, SEQ ID NO:65;R372K/R377K, SEQ ID NO:66; and Sema3A short peptide, SEQ ID NO:67.

FIGS. 36A-B shows expression of NRP1 in pancreatic cancer cell lines andbreast cancer cell lines. (FIG. 36A) shows expression of NRP1 in somepancreatic cancer cell lines (BxPC-3, CFPAC-1, Panc-1 and SU8686) andpancreatic epithelial cells as analyzed by RT-PCR analysis. (FIG. 36B)shows expression of NRPI in breast cancer cell lines (BT-20, MDA-MB-231,SKBR-3, TD47D and ZR-75-1) as analyzed by RT-PCR analysis. In all RT-PCRanalysis, β-actin was used as positive control.

FIG. 37 shows that Sema3A-nLytic induces Annexin V-positive expressionin cancer cells. PE cells (upper panel) and BxPC-3 cell (lower panel)were incubated with Sema3A-nLytic peptide (5 μM) at 37° C. for threehours, and after six hours, analyzed for Annexin V labeling bydual-color flow cytometry.

FIG. 38A shows a graph of cell-killing effect of Sema3A(aa363-377)-kLytic or kLytic alone to cancer cells. Four pancreaticcancer cell lines ((a) BxPC-3, (b) CFPAC-1, (c) Panc-1 and (d) SU8686)were cultured with various concentrations of Sema3A(aa363-377)-kLytic(quadrangle) or kLytic (triangle) for 48 hours, and cytotoxic activitywas assessed using WST-8 reagent. The vertical axis shows cell viability(%) and the horizontal axis shows peptide concentration (μM).

FIG. 38B shows a graph of cell-killing effect of kLytic (quadrangle) orSema3A(aa363-377)-kLytic (triangle) to normal cells. Three normal celllines ((a) PE, (b) MRC-5 and (c) human normal hepatocyte) were culturedwith various concentrations of Sema3A(aa363-377)-kLytic or kLytic for 48hours, and cytotoxic activity was assessed using WST-8 reagent. Thevertical axis shows cell viability (%) and the horizontal axis showspeptide concentration (μM).

FIG. 39A shows expression of neuropilin-1 in some pancreatic cancer celllines (BxPC-3, Panc-1, SU8686 and CFPAC-1) and pancreatic epithelialcells as estimated by RT-PCR analysis. For all RT-PCR analyses, GAPDHwas used as positive control.

FIG. 39B shows determination of expression of neuropilin-1 by real-timePCR. For each of the cells shown in the graph of FIG. 39A, expression ofneuropilin-1 was determined using real-time PCR. All controls wereanalyzed using GAPDH.

FIG. 40 shows the results of incubating human pancreatic cancer cellline SU8686 with Sema3A(363-377)-kLytic peptide (10 μM) at 37° C. forthree hours and performing dual-color flow cytometry analysis forAnnexin V labeling. These results indicate that Sema3A(363-377)-kLyticalso induces Annexin V-positive expression to cancer cells.

FIG. 41 shows cytotoxic activity of VEGFR2-lytic peptide to cancer cellsand normal cells. Five cancer cell lines (OE19 (white triangle), T47D(black triangle), Bxpc3 (white rhomboid), U937 (black rhomboid) andLNCaP (x)) and two normal cell lines (HEK293 (white circle) and MRC-5(black circle)) were cultured with various concentrations (0 to 20 μM)of VEGFR2-lytic peptide for 72 hours, and cytotoxic activity wasassessed using WST-8 reagent. The vertical axis shows cell viability (%)and the horizontal axis shows peptide concentration (μM). The assay wasrepeated three times, and the results are represented as mean oftriplicate measurements±SD (bar). It is seen that VEGFR2-lytic peptidehas cytotoxic activity specific for cancer cells.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, regarding the present invention, embodiments of theinvention will be explained. It should be understood that throughout thepresent specification singular expressions (corresponding articles,adjectives and the like in other languages, such as “a,” “an” and “the”in English) also include concepts in plural form, unless particularlymentioned. Further, it should be understood that the terms used hereinare used in the meaning normally used in the art, unless particularlymentioned. Thus, unless defined otherwise, all technical terms andscientific technology terms used herein have the same meaning asgenerally understood by those skilled in the art to which the presentinvention belongs. In the case of contradiction, the presentspecification (including definitions) precedes.

DEFINITIONS OF TERMS

Hereinafter, definitions of terms particularly used herein are listed.

As used herein, “epidermal growth factor (EGF)” refers to a growthfactor belonging to EGF superfamily. In terms of history, arepresentative example of EGF is a polypeptide having a molecular weightof about 6,000 and consisting of 53 amino acids, which has been reportedas a pharmacologically active substance present in the submandibulargland of a male mouse and which has three disulfide bonds composed ofsix cysteines in a molecule. For the first time, it was believed to actspecifically on the growth of epidermal cells, but it also acts onnon-epidermal cells, and exhibits various biological activities. As EGF,genes of the following GenBank accession number or the like can beincluded as representative examples: GenBank#NM_(—)001963 (human) orEntrez Gene ID 1950.

As used herein, “epidermal growth factor receptor” and “EGF receptor”(EGFR) are interchangeably used, and refer to a receptor, of which aligand is EGF. EGF receptor was found as v-erbB, one of viral cancergenes present in a genome of avian erythroblastosis virus (AEV). Acorresponding gene c-erbB1 present in a human genome is an EGFR gene.The structure thereof is as described below.

Specifically, an EGF receptor is composed of a single polypeptide. TheN-terminal side continues to an extracellular region having aligand-binding site, and forms a single-spanning transmembrane region.The C-terminal side forms an intracellular region having tyrosine kinaseactivity. The receptor is associated via binding of a ligand, resultingin activation of tyrosine kinase. A human EGF receptor is atransmembrane glycoprotein having a molecular mass of about 170 kDa,among which an extracellular region causing association of a receptor ina ligand-dependent manner is a glycoprotein of about 95 kDa (Ogiso H, etal., 2002, Cell, 110:775-87). Binding of EGF or TGF-α to EGFR activatesa signal transduction pathway to cause cellular growth. Dimerization,high-order structural change and internalization of an EGFR moleculefunction to transduce intracellular signal to cause cellular growthcontrol (G. Carpenter and S. Cohen, 1979, Ann. Rev. Biochem.,48:193-216). Genetic change which influences on control of a growthfactor receptor functions to or leads to over-express a receptor and/ora ligand, thereby causing cellular growth (M.-J. Oh et al., 2000, Clin.Cancer Res., 6:4760-4763). GenBank# is NM_(—)005228 (human). As EGFR, inaddition, the gene of the following GenBank accession number or the likecan be included as a representative example: Entrez Gene ID 1956.

As used herein, “target-binding” peptide or “target-binding” sequencerefers to a peptide or sequence which binds to a target (for example,EGF receptor or other target). For example, a binding sequence specificfor a receptor with high expression in cancer cells can be included asan example thereof.

As used herein, “a binding sequence specific for a receptor with highexpression in cancer cells” refers to a sequence specifically binding toa receptor which expresses highly in cancer cells. A receptor for whichhigh expression is observed in cancer cells as specifically definedbelow includes peptide sequences which respectively bind specifically toa growth factor receptor involved in cellular growth, a receptorinvolved in angiogenesis, a cytokine/chemokine receptor, and the like.By phage display technique, three-dimensional structure analysis or thelike, specific binding sequences are found.

As used herein, “EGF receptor-binding peptide (EB peptide)” or “EGFreceptor-binding sequence (EB sequence)” refers to a peptide or sequencebinding to EGF receptor.

Typically, the following can be included as examples of EGFreceptor-binding sequences.

1) YHWYGYTPQNVI (SEQ ID NO: 7; wherein H may be substituted with R orK). These modified sequences are sequences screened from a peptidelibrary by phage display technique, and have never been published inmutation analysis or the like conducted so far. They are mutants inwhich H having a plus charge has been substituted with R or K, whilepaying attention to an amino acid having a charge believed to beimportant in receptor-ligand bond, and are expected to have asignificant effect in the present invention.

Herein, when not particularly mentioned, “lytic peptide moiety,”“cytotoxic peptide” and “cytotoxic sequence” are used interchangeablywith “cytolytic peptide (sequence)” or “cell membrane-lytic peptide(sequence).” They have a peptide which may lyse a cell membrane.Typically, examples thereof include those which are composed of an aminoacid having a plus charge [K, R or (H)] and a hydrophobic amino acid [L,A, F or the like] and which have a 10- to 30-amino acids sequence withan amphipathic helix structure. Representative peptide sequences asthose which act on a cancer cell membrane to exhibit cell-killing effectand which may be particularly used in the present invention aredescribed below:

KLLLKLLKKLLKLLKKK (SEQ ID NO: 48; specifically SEQ ID NO: 1, 27 or thelike): cell membrane-lytic, FLKLLKKLAAKLF (SEQ ID NO: 11): antibacterialpeptide derivative, cell membrane potential-disintegrating,RLLRRLLRRLLRRLLRRLLRRLLR (SEQ ID NO: 12) and RLLRRLLRRLLRK (SEQ ID NO:13): antibacterial peptide derivative, cell membrane-lytic and nucleicacid-biding, KLAKLAKKLAKLAK (SEQ ID NO: 4): mitochondrial membranedisintegrating.

Thus, from such information, it can be recognized that representativecytotoxic peptide is a 10- to 30-amino acids sequence composed of anamino acid having a plus charge [K, R or (H)] and a hydrophobic aminoacid [L, A, F or the like], and having amphiphatic helix structure.

It revealed that use of KLLLKLLKKLLKLLKKK (SEQ ID NO: 1; the underlinedletters represent D-amino acids) attains a significant effect incomparison with LKLLKKLLKKLLKLL-NH₂ (SEQ ID NO: 41; the underlinedletters represent D-amino acids) known as the original sequence, on thefollowing points: for example, it revealed that combination of anEGFR-binding sequence before the sequence results in synergistic effectin a cell-killing activity in a cancer cell-specific manner. It alsorevealed that more selectivity with normal cells is achieved.Hybridization with various target proteins is possible. Thus, it isunderstood that further targeting to cancer cells is possible.

Furthermore, it revealed that a peptide referred to as nLytic(LLKLLKKLLKKLLKL; SEQ ID NO: 45; the underlined letters representD-amino acids) attains a significant effect on the following points: asit revealed that selectivity with normal cells are achieved, it isbelieved that the toxicity is low. It also revealed that combinationwith Sema3A peptide results in synergistic effect in a cell-killingactivity in a cancer cell-specific manner. Thus, it is understood thatfurther targeting to cancer cells is possible.

As used herein, “spacer” refers to a portion which forms a chemical bondbetween molecules of chain macromolecules, like bridging. Representativeexamples of spacer include a 0- to 5-amino acids sequence consisting ofG and P. Herein, particularly, a spacer may intervene and may be boundbetween a lytic peptide and an EGF receptor-binding peptide. Examples ofsuch a spacer include, for example, GGG, GG, G, PP and GPG. A spacer isnot essential and may be absent, but in the present invention, such aspacer is preferably included.

As used herein, “peptidetoxin” refers to an anticancer-targeted peptidewhich has cytotoxicity and cell-killing ability. Peptidetoxin of thepresent invention can include peptides formed by combining cytotoxicmoiety which corresponds to an explosive portion and a moiety assignedfor specificity for cancer cells, which corresponds to a warhead portion(for example, a peptide/sequence binding specifically to a receptorwhich expresses highly in cancer cells). Regarding “explosive portion,”in the actual circumstance, a cell membrane-lytic sequence is preferred,and regarding “warhead portion,” any sequence that binds specifically toa protein (particularly receptor) which highly expresses in cancer cellsis possible. For example, “cancer cell membrane-lytic peptidetoxin”composed of a binding sequence specific for a receptor with highexpression in cancer cells, spacer and a cancer cell membrane-lyticsequence can be included as a representative example.

Methods of production and use of “cancer cell membrane-lyticpeptidetoxin” composed of a binding sequence specific for a receptorwith high expression in cancer cells, spacer and cancer cellmembrane-lytic sequence are described below. Herein, any bindingsequence specific for a receptor with high expression in cancer cells,any spacer, and any cancer cell membrane-lytic sequence can be combinedarbitrarily. Methods of production and use thereof are specificallydescribed below.

(Method of Production)

For peptidetoxins formed of a number of combinations of warhead andexplosive sequences, which allow early individuated therapy of cancer,chemical synthesis capable of providing them in a short period issuitable, but a method is also possible in which peptidetoxins areforcibly expressed by genetic recombination and then purified.

(Method of Use)

Regarding a cancer cell to be treated, profile of a protein which highlyexpresses on cell surface and sensitivity to damage of the cancer cellagainst the explosive peptide are investigated. Based on the results,the warhead and explosive are selected, and a peptidetoxin optimal forthe cancer cell is designed. A tailor-made peptidetoxin obtained bychemical synthesis or the like is combined with DDS such asatelocollagen depending on necessity, and is topically or systemicallyadministered for treatment.

The cell membrane-lytic sequence used by the present inventors, whenused alone, contact for a long time is necessary for exhibiting acell-killing effect on cancer cells, and the cell-killing effect is alsomild. However, when the cell membrane-lytic sequence is conjugated witha cancer cell-targeted sequence to be chimerized, the resulting sequencebinds preferentially to cancer cells in which the target molecules ofthe sequence are highly expressed. Thus, contact time can be reduced,and the cell-killing effect is also enhanced. Actually, in chimerizationof binding sequence such as IL4 receptor, her2 or the like, enhancementof cell-killing effect and reduction of contact time were confirmed.Accordingly, it can be rationally expected that even other similarchimeric sequences attain similar effects of enhancement of cell-killingeffect and reduction in contact time.

Regarding peptidetoxins which allow such individuated therapy at earlystage, combination of warhead and explosive can lead to a landmarktherapy for patients with cancer. Thus, the new concept of peptidetoxinsof the present invention is important in that it provides the maximumadvantage for patients with cancer.

A chimeric peptide or peptidetoxin of the present invention should benoted in that it attained cancer cell targeting, enhancement ofcell-killing effect and instantaneous cell-killing effect with respectto cancer cells as compared to a cell membrane-lytic peptide alone, as aresult of chimerization, even in comparison with the closest prior artin the circumstance where various types of chimeric peptides have beengranted a patent.

Sequences of a portion assigned for cell membrane permeability, whichcan be used herein are as described below. Common feature includes, forexample, content of a large amount of amino acids having a charge.

As used herein, “cell-permeable peptide” refers to a peptide capable ofpassing a cell membrane to invade inside the cell. Whether or not apeptide is “a cell-permeable peptide” can be evaluated by the followingtest. Specifically, herein, regarding Antp, as described in knownmethods (see Derossi et al., J. Biol. Chem. 1996, 271, 18188-18193), itis possible to add a biotinized Antp peptide to a cell, subsequently adda compound chemically labeled with streptavidin, and confirmlocalization in the cell using fluorescence microscope. Alternatively,it is possible to similarly confirm localization of an antibody whichhas been reacted with a streptavidin-binding antibody and thenchemically labeled in the same manner using fluorescence microscope,thereby confirming invasion inside the cell.

Examples of cell-permeable peptides can include, for example,RQIKIQFQNRRMKWKK (Antp; SEQ ID NO: 58) which is Antennapedia homeoboxsequence, YGRKKRRQRRR (TAT; SEQ ID NO: 23), RRRRRRRRRRR (SEQ ID NO: 24)and the like. Typically, the structure thereof can include, for example,Gene ID 155871 (TAT protein per se). As cell-permeable peptide, inaddition, a gene of the following GenBank accession number or the likecan be a representative example: NP_(—)057853; Tat [humanimmunodeficiency virus 1, amino acid sequence] MEPVDPRLEP WKHPGSQPKTACTNCYCKKC CFHCQVCFIT KALGISYGRK KRRQRRRAHQ NSQTHQASLS KQPTSQPRGD PTGPKE1 (SEQ ID NO: 57).

Examples of a sequence of a portion assigned for specific inhibition ofcancer cell growth in the cytoplasm, which can be used herein includeKAYARIGNSYFK (TPR; SEQ ID NO: 59) which is HSP90 TPR domain-bindingpeptide, and the like.

Representatives cytotoxic peptides used herein can include cellmembrane-lytic peptide, cell membrane potential-destabilizing peptide,cell membrane-lytic and nucleic acid-binding peptide and mitochondrialmembrane-disintegrating peptide.

As used herein, “cell membrane-lytic peptide” refers to a peptidecomposed of an amino acid having a plus charge [K, R or (H)] and ahydrophobic amino acid [L, A, F or the like] and having a 10- to30-amino acids sequence with an amphipathic helix structure, whichdisintegrates a cell membrane from outside to exhibit a cell-killingeffect. Representative specific examples include: a peptide having a 10-to 20-amino acids sequence consisting only of K and L, wherein the aminoacids are L-, D- or D,L-mix amino acids, such as KLLLKLLKKLLKLLKK (SEQID NO: 48; specifically SEQ ID NO: 1, 27 or the like).

As used herein, “cell membrane potential-destabilizing peptide” refersto a peptide composed of an amino acid having a plus charge [K, R or(H)] and a hydrophobic amino acid [L, A, F or the like] and having a 10-to 30-amino acids sequence, which forms a pore on a cell membrane fromthe outside, destabilizes the cell membrane potential, disintegrate thecell membrane, and exhibits a cell-killing effect. Representativespecific examples include FLKLLKKLAAKLF (SEQ ID NO: 11) and the like.

As used herein, “cell membrane-lytic and nucleic acid-binding peptide”refers to a peptide composed of an amino acid having a plus charge [K, Ror (H)] and a hydrophobic amino acid [L, A, F or the like] and having a10- to 30-amino acids sequence with an amphipathic helix structure,which disintegrates a cell membrane from outside, invades inside thecell, binds to a nucleic acid and induces cell death. Representativespecific examples include a peptide having a 10- to 20-amino acidssequence consisting only of K and L, wherein the amino acids are L-, D-,or D,L-mix amino acids, such as RLLRRLLRRLLRRLLRRLLR (SEQ ID NO: 12),RLLRRLLRRLLRK (SEQ ID NO: 13) and the like.

As used herein, “mitochondrial membrane-disintegrating peptide” refersto a peptide composed of an amino acid having a plus charge [K, R or(H)] and a hydrophobic amino acid [L, A, F or the like] and having a 10-to 30-amino acids sequence with an amphipathic helix structure, whichdisintegrates a mitochondrial membrane to induce cell death only uponsuccessful invasion inside the cell. Representative specific examplesinclude KLAKLAKKLAKLAK (SEQ ID NO: 4) and the like.

Preferred examples of cytotoxic peptides include a peptide composed ofan amino acid having a plus charge [K, R or (H)] and a hydrophobic aminoacid [L, A, F or the like] and having a 10- to 30-amino acids sequencewith an amphipathic helix structure. Preferably, the amino acid having aplus charge may be K, R or H, and the hydrophobic amino acid may be I,L, V, A or F.

The hydrophobic amino acid used in the present invention isKLLLKLLKKLLKLLKKK, wherein each amino acid is L- or D-amino acid and theunderlined letters represent D-amino acids.

As a matter clarified by the present invention, it revealed that achimeric peptide in which a receptor-binding peptide, such as EGFR, hasbeen conjugated is advantageous to have an amphipathic helix structure,preferably α-helix structure. Thus, also in a case of constructing achimeric peptide including other receptor-binding peptide and cytotoxicpeptide, it is advantageous that such a peptide has an amphipathic helixstructure, preferably α-helix structure. It is understood that thesematters can be also deduced from the results shown in FIG. 1D.Specifically, it has been demonstrated that EB-lytic peptide weaklybound to small unilamellar vesicles (SUVs) composed ofphosphatidylcholine (PC), which is the dominant lipid species on thesurface of normal cell membranes, and was not well structured with thePC liposome, while this EB-lytic peptide was capable of binding to SUVscontaining phosphatidylserine (PS), which is exposed specifically oncancer cell membranes, and conformed to a partial helical structure ascharacterized by double minima at 209-210 and 222 nm. It has revealedthat, EB-original lytic peptide was capable of binding strongly to bothPC and PC/PS liposomes, and conformed to helical structures (FIG.1D(c)). It is indicated that the chimeric peptide newly designed in thepresent invention has a selectivity to PS-containing membranes andconforms to a helical structure which is supposed to be essential formaking a pore on the cell surface (Papo, N. & Shai, Y. New Lyticpeptides based on the D,L-amphipathic helix motif preferentially killtumor cells compared to normal cells. Biochemistry 42, 9346-9354(2003)). Thus, these data can be a reference and help in designing otherforms of the lytic peptide.

Examples of such include a sequence selected from the group consistingof SEQ ID NO: 2, 14, 21, 42 and 43 or a modified sequence thereof. Here,modified sequence includes a sequence which includes one or severalamino acid substitutions, additions or deletions, preferablyconservative substitutions, of the sequence specifically described here.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 15 or a modified sequence thereof.Here, modified sequence includes a sequence which includes one orseveral amino acid substitutions, additions or deletions, preferablyconservative substitutions, of the sequence specifically described here.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 16 or a modified sequence thereof.Here, modified sequence includes a sequence which includes one orseveral amino acid substitutions, additions or deletions, preferablyconservative substitutions, of the sequence specifically described here.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 17 or a modified sequence thereof.Here, modified sequence includes a sequence which includes one orseveral amino acid substitutions, additions or deletions, preferablyconservative substitutions, of the sequence specifically described here.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 18 or 44 or a modified sequencethereof. Here, modified sequence includes a sequence which includes oneor several amino acid substitutions, additions or deletions, preferablyconservative substitutions, of the sequence specifically described here.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 19 or a modified sequence thereof.Here, modified sequence includes a sequence which includes one orseveral amino acid substitutions, additions or deletions, preferablyconservative substitutions, of the sequence specifically described here.

In one embodiment, a chimeric peptide of the present invention has thesequence set forth in SEQ ID NO: 20, 46 or 47 or a modified sequencethereof. Here, modified sequence includes a sequence which includes oneor several amino acid substitutions, additions or deletions, preferablyconservative substitutions, of the sequence specifically described here.

Regarding a modified sequence of them, those skilled in the art cancarry out modification such that the sequence has a preferredamphipathic (a) helical structure, based on the description herein.Furthermore, for example, regarding one amino acid substitution, it hasbeen demonstrated that an amphipathic (a) helical structure is retainedin a chimeric peptide related to EGF-R. It is understood that otherchimeric peptide is also expected to retain the amphipathic (a) helicalstructure. Furthermore, it is understood that other modifications suchas substitution of a plurality of amino acids can be appropriatelycarried out by those skilled in the art with reference to thedescriptions in the Examples, and the like.

In another preferred embodiment, as a cancer cell growth-inhibitingpeptide which acts intracellularly, a peptide having the sequence ofRQIKIQFQNRRMKWKKKAYARIGNSYFK (SEQ ID NO: 25) is used. Alternatively, apeptide referred to as TAT (YGRKKRRQRRR) (SEQ ID NO: 23) may also beused. It is known that a sequence in which 11 Rs are linked, RRRRRRRRRRR(SEQ ID NO: 24), permeates cells, and such a sequence can also be used.Furthermore, those skilled in the art can appropriately determinepreferred combination of cell-permeable peptide and TPR domain-bindingpeptide. Preferably, combination with Antp can be used.

As used herein, “chimeric peptide” refers to a peptide formed of two ormore moieties (peptides) of different genotypes. It is also referred toas a fusion protein. It is used for studying function of a domain of aprotein or detecting expression of a protein of interest.

As used herein, “similar amino acid” refers to an amino acid which is ina relationship of conservative substitution, and the following aminoacids correspond:

A: G, I, V, L

C: M (S-containing amino acid)

D: N, Q or E

E: Q, N or D

F: Y, A or the like

G: A

H: W, R, K or the like

I: A, L, V, (G)

K: R, H

L: A, I, V, (G)

M: S or the like

N: D, E or Q

P: HyP

Q: E, N or D

R: K, H

S: T, Y

T: S, Y

V: I, L, A, (G)

W: H

Y: F, S, T.

Substitutions between these amino acids are also referred to as“conservative substitution” herein.

Herein, amino acids which are frequently found in an EGFreceptor-binding peptide refer to those which are found frequently invarious EGF receptor-binding peptides.

Typically, amino acids in a relationship of conservative substitutionare included. Or, the following amino acids correspond:

the peptide having an amino acid sequence X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂(SEQ ID NO: 8), wherein:

X₁ is Y or an amino acid similar thereto;

X₂ is H or an amino acid similar thereto;

X₃ is W or an amino acid similar thereto;

X₄ is Y or an amino acid similar thereto;

X₅ is G or an amino acid similar thereto;

X₆ is Y or an amino acid similar thereto;

X₇ is T or an amino acid similar thereto;

X₈ is P or an amino acid similar thereto;

X₉ is Q or an amino acid similar thereto;

X₁₀ is N or an amino acid similar thereto;

X₁₁ is V or an amino acid similar thereto; and

X₁₂ is I or an amino acid similar thereto.

Preferably, X₁ is Y, or an amino acid similar thereto having an OH groupor an aromatic group;

X₂ is H, or an amino acid similar thereto having a plus charge;

X₃ is W, or an amino acid similar thereto having an aromatic group;

X₄ is Y, or an amino acid similar thereto having an OH group;

X₅ is G, or an amino acid similar thereto having an aliphatic sidechain;

X₆ is Y, or an amino acid similar thereto having an OH group;

X₇ is T, or an amino acid similar thereto having an OH group;

X₈ is P or an imino acid-type amino acid similar thereto;

X₉ is Q or an amide-type amino acid similar thereto;

X₁₀ is N, or an amino acid similar thereto having an OH group;

X₁₁ is V, or an amino acid similar thereto having an aliphatic sidechain; and

X₁₂ is I, or an amino acid similar thereto having an aliphatic sidechain.

More preferably, X₁ is Y, or an amino acid similar thereto which is S, Hor F;

X₂ is H, or an amino acid similar thereto which is R or K;

X₃ is W, or an amino acid similar thereto which is Y, F or H;

X₄ is Y, or an amino acid similar thereto which is S, H or F;

X₅ is G, or an amino acid similar thereto which is A, V, I or L;

X₆ is Y, or an amino acid similar thereto which is S, H or F;

X₇ is T, or an amino acid similar thereto which is S, H or F;

X₈ is P, or an amino acid similar thereto which is hydroxyl proline;

X₉ is Q, or an amino acid similar thereto which is N;

X₁₀ is N, or an amino acid similar thereto which is S, H or F;

X₁₁ is V, or an amino acid similar thereto which is G, A, L or I; and

X₁₂ is I, or an amino acid similar thereto which is G, A, V or L.

A sequence in which X₂ is H or an amino acid similar thereto, which is Ror K (for example, YRWYGYTPQNVI (SEQ ID NO: 9) or YKWYGYTPQNVI (SEQ IDNO: 10)) is preferred, because enhanced activity was found.

Herein, when a receptor-binding peptide used is interleukin 4 (IL-4)receptor-binding peptide, the amino acid sequence KQLIRFLKRLDRN (SEQ IDNO: 26) or a modified sequence thereof may be used. Here, the modifiedsequence can be modified in the same manner as the EGFR-binding peptide.In this modification, Thorsten Hage et al., Cell. 1999, vol. 97, No. 2,pp. 271-81 can be referenced. This literature is incorporated herein asa reference.

Herein, when a receptor-binding peptide used is interleukin 13 (IL-13)receptor-binding peptide, the amino acid sequence KDLLLHLKKLFREGQFN (SEQID NO: 28) or a modified sequence thereof may be used. Here, themodified sequence can be modified in the same manner as the EGFR-bindingpeptide. In this modification, Yuichiro Yoshida et al., Biochem BiophysRes Commun. 2007, vol. 358, No. 1, pp. 292-297 can be referenced. Thisliterature is incorporated herein as a reference.

Herein, when a receptor-binding peptide used is neuropilinreceptor-binding peptide, the amino acid sequence NYQWVPYQGRVPYPR (SEQID NO: 29) or a modified sequence thereof may be used. Here, themodified sequence can be modified in the same manner as the EGFR-bindingpeptide. In this modification, Alexander Antipenko et al., Neuron. 2003,vol. 39, No. 4, pp. 589-598 can be referenced. This literature isincorporated herein as a reference. These literatures merely presentproblems in protein formulations such as immunotoxins, and do notprovide a chimeric peptide as in the present invention.

Herein, when a receptor-binding peptide used is human epidermal growthfactor receptor type 2 (HER2)-binding peptide, the amino acid sequenceYCDGFYACYMDV (SEQ ID NO: 30), LLGPYELWELSH (SEQ ID NO: 52),ALVRYKDPLFVWGFL (SEQ ID NO: 53), KCCYSL (SEQ ID NO: 54),WTGWCLNPEESTWGFCTGSF (SEQ ID NO: 55), DTDMCWWWSREFGWECAGAG (SEQ ID NO:56) or a modified sequence thereof may be used. Here, the modifiedsequence can be modified in the same manner as the EGFR-binding peptide.In this modification, Valeria R. Fantin et al., Cancer Res. 2005, vol.65, No. 15, pp. 6891-6900 can be referenced. This literature isincorporated herein as a reference.

Herein, when a receptor-binding peptide used is vascular epithelialgrowth factor receptor 1 (VEGFR1)-binding peptide, the amino acidsequence WHSDMEWWYLL (SEQ ID NO: 31) or a modified sequence thereof maybe used. Here, the modified sequence can be modified in the same manneras the EGFR-binding peptide. In this modification, Kimberly J. Petersonet al., Analytical Biochemistry 2008, vol. 378, No. 1, pp. 8-14(regarding VEPNCDIHVMWEWECFERL-NH2 (SEQ ID NO: 32)) and Borlan Pan etal., J. Mol. Biol. 2002, vol. 316, No. 3, pp. 769-787 (regardingGGNECDAIRMWEWECFERL (SEQ ID NO: 33)) can be referenced. Theseliteratures are incorporated herein as references.

Herein, when a receptor-binding peptide used is Transferrin Receptor(TfR)-binding peptide, the amino acid sequence THRPPMWSPVWP (SEQ ID NO:34) or a modified sequence thereof may be used. Here, the modifiedsequence can be modified in the same manner as the EGFR-binding peptide.In this modification, Jae H. Lee et al., Eur. J. Biochem. 2001, vol.268, pp. 2004-2012 can be referenced. This literature is incorporatedherein as a reference.

In addition, a receptor-binding peptide may be a fibroblast growthfactor receptor (FGFR)-binding peptide which is MQLPLAT (SEQ ID NO: 5)or AAVALLPAVLLALLAP (SEQ ID NO: 6); neuropilin 1 (NRP1)/vascularendothelial growth factor receptor 2 (VEGFR2)-binding peptide which isATWLPPR (SEQ ID NO: 36); ephrin B1 (EphB1)-binding peptide which is EWLS(SEQ ID NO: 37); ephrin B2 (EphB2)-binding peptide which is SNEW (SEQ IDNO: 38); interleukin-11 receptor (IL11R)-binding peptide which isCGRRAGGSC (cyclic) (SEQ ID NO: 22); a glucose-regulating protein 78(GRP78)-binding peptide which is WDLAWMFRLPVG (SEQ ID NO 39) orCTVALPGGYVRVC (cyclic) (SEQ ID NO: 40); a prostate-specific membraneantigen (PSMA)-binding peptide which is CQKHHNYLC (SEQ ID NO: 35); or amodified sequence thereof. Here, the modified sequence can be modifiedin the same manner as the EGFR-binding peptide. In this modification,the following literatures can be referenced: for FGFR (MQLPLAT (SEQ IDNO: 5)), Fukuto Maruta et al., Cancer Gene Therapy. 2002, vol. 9, pp.543-552; for FGFR (AAVALLPAVLLALLAP (SEQ ID NO: 6)), Akiko Komi et al.,Exp. Cell Res. 2003, Vol. 283, No. 1, pp. 91-100; for NRP1/VEGFR2(ATWLPPR (SEQ ID NO: 36)), Loraine Tirand et al., J. Control Release.2006, vol. 111, pp. 153-164; for EphB1 and EphB2, Mitchell Koolpe etal., J. Biol. Chem. 2005, vol. 280, No. 17, pp. 17301-11; for IL11R,Amado J. Zurita et al., Cancer Res. 2004, vol. 64, pp. 435-439; forGRP78 (WDLAWMFRLPVG (SEQ ID NO: 39)), Marco A. Arap et al., Cancer Cell.2004, vol. 6, pp. 275-284; for GRP78 (CTVALPGGYVRVC (SEQ ID NO: 40) andthe like), Ying Liu et al., Mol. Pharmaceutics. 2007, Vol. 4, No. 3, pp.435-447, Hardy B et al., Therapeutic angiogenesis of mouse hind limbischemia by novel peptide activating GRP78 receptor on endothelialcells. Biochemical Pharmacology 75,891-899, 2008; and for PSMA, KaushalRege et al., Cancer Res. 2007, vol. 67, No. 13, pp. 6368-6375. Theseliteratures are incorporated herein as references.

These substitutions may be used alone or in combination of more thanone. It is understood that any combination of these preferredsubstitutions may be effective, because enhancement of the effect wasfound as a result of these substitutions. It is understood that eitherone or a plurality of these mutations may be introduced. This isbecause, although not desired to be restricted by a theory, when it isunderstood that a mutation is permitted, it is understood that thethree-dimensional structure and activity in interaction with abiological target and the like of the original active type is retainedor enhanced, and thus it is expected that combination of a plurality ofthem also has a similar effect. In the case of the present invention,regarding change in activity as a result of one amino acid substitution,it is predicted that interaction with a partner protein thereof wasinfluenced, but from a viewpoint of anticancer activity, the finalpurpose of the present invention, the activity was retained. Thus, itcan be expected that a similar effect is obtained even in the case ofcombination of these amino acid substitutions.

As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide”are used in the same meaning in the present specification and refer toan amino acid polymer having any length. This polymer may be straight,branched or cyclic. An amino acid may be a naturally occurring ornon-naturally occurring amino acid, and may be a modified amino acid.These terms may encompass those assembled with a complex of a pluralityof polypeptide chains. These terms further encompass a naturallyoccurring or artificially modified amino acid polymer. Examples of sucha modification include, for example, formation of a disulfide bond,glycosylation, lipidation, acetylation, phophorylation, or any othermanipulation or modification (for example, conjugation with a labelcomponent). The definition also encompasses, for example, a polypeptideincluding one or more analog(s) of amino acid (including, for example,an unnatural amino acid and the like), peptide-like compounds (forexample, peptoid) and other modifications known in the art.

As used herein, “amino acid” may be either naturally occurring ornon-naturally occurring amino acid, as long as it satisfies the purposeof the present invention.

As used herein, “nucleic acid” is used interchangeably with gene, cDNA,mRNA, oligonucleotide and polynucleotide. A particular nucleic acidsequence also encompasses “splice variant.” Similarly, a particularprotein encoded by a nucleic acid implicitly encompasses any proteinencoded by a splice variant of the nucleic acid. As suggested by thename, “splice variant” is a product of alternative splicing of a gene.After transcription, the first nucleic acid transcript may be spliced sothat a different (other) nucleic acid splice product encodes a differentpolypeptide. A production mechanism of a splice variant is changed, andalternative splicing of exon is included. Other polypeptide derived fromthe same nucleic acid by read through transcription is also encompassedin this definition. Any product of splicing reaction (including spliceproduct of recombinant form) is encompassed in this definition. Or,allelic gene mutant is also included within this scope.

As used herein, “polynucleotide,” “oligonucleotide” and “nucleic acid”is used in the same meaning and refer to a nucleotide polymer having anylength. These terms may encompass “oligonucleotide derivative” or“polynucleotide derivative.” “Oligonucleotide derivative” or“polynucleotide derivative” refers to oligonucleotide or polynucleotidewhich includes a nucleotide derivative or in which a bond betweennucleotides is different from usual, and these terms are usedinterchangeably. Specific examples of such oligonucleotide include, forexample, 2′-O-methyl-ribonucleotide, oligonucleotide derivative obtainedby converting phosphodiester bond in the oligonucleotide intophosphorothioate bond, oligonucleotide derivative obtained by convertingphosphodiester bond in the oligonucleotide into N3′-P5′ phosphoroamidatebond, oligonucleotide derivative obtained by converting ribose andphosphodiester bond in the oligonucleotide into peptide nucleic acidbond, oligonucleotide derivative obtained by substituting uracil in theoligonucleotide with C-5 propynyl uracil, oligonucleotide derivativeobtained by substituting uracil in the oligonucleotide with C-5 thiazoluracil, oligonucleotide derivative obtained by substituting cytosine inthe oligonucleotide with C-5 propynyl cytosine, oligonucleotidederivative obtained by substituting cytosine in the oligonucleotide withphenoxazine-modified cytosine, oligonucleotide derivative obtained bysubstituting ribose in DNA with 2′-O-propylribose, oligonucleotidederivative obtained by substituting ribose in the oligonucleotide with2′-methoxyethoxy ribose, and the like. Unless otherwise indicated, it isintended that a particular nucleic acid sequence encompasses anexpressly described sequence and a conservatively modified variantthereof (for example, variant with substitution of degeneracy codon) anda complementary sequence. Specifically, a variant with substitution ofdegeneracy codon may be achieved by producing a sequence in whichposition 3 of one or more selected (or all) codons has been substitutedwith mix base and/or deoxyinosine residue (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

As used herein, “nucleotide” may be either naturally occurring ornon-naturally occurring nucleotide, as long as the desired function isretained.

As used herein, “search” refers to utilizing a nucleic acid basesequence to find other nucleic acid base sequence having a particularfunction and/or property by electronic or biological method or othermethod. Electronic search includes, but is not limited to, BLAST(Altschul et al., J. Mol. Biol. 215:403-410 (1990)), FASTA (Pearson &Lipman, Proc. Natl. Acad. Sci., USA 85:2444-2448 (1988)), Smith andWaterman method (Smith and Waterman, J. Mol. Biol. 147:195-197 (1981)),and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol.48:443-453 (1970)). Biological search includes, but is not limited to,stringent hybridization, microarray formed by attaching genomic DNA onnylon membrane or the like, microarray formed by attaching genomic DNAto a glass plate (microarray assay), PCR, in situ hybridization, and thelike. Herein, it is intended that a gene used in the present invention(for example, HSP90 or the like) should include corresponding genesidentified by such electronic search or biological search.

Herein, a nucleic acid sequence which hybridizes to a particular genesequence can also be used, as long as it has a function. Here,“stringent conditions” for hybridization refers to conditions underwhich a complementary strand of a nucleotide chain having similarity orhomology to a target sequence preferentially hybridizes to a targetsequence and a complementary strand of a nucleotide chain withoutsimilarity or homology does not substantially hybridize. “Complementarystrand” of a nucleic acid sequence refers to a nucleic acid sequencewhich pairs based on a hydrogen bond between bases of the nucleic acid(for example, T to A and C to G). Stringent conditions aresequence-dependent, and vary in different situations. A longer sequencehybridizes specifically at a higher temperature. Generally, stringentconditions are selected at about 5° C. lower than thermal meltingtemperature (T_(m)) for a particular sequence at a defined ionicstrength and pH. T_(m) is a temperature at which 50% of nucleotidescomplementary to a target sequence hybridize to a target sequence atequilibrium state at a defined ionic strength, pH and nucleic acidconcentration. “Stringent conditions” are sequence-dependent, and varydepending on various environmental parameters. General guideline ofhybridization of nucleic acid are found in Tijssen (Tijssen (1993),Laboratory Techniques In Biochemistry And MolecularBiology-Hybridization With Nucleic Acid Probes Part I, Chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assay”, Elsevier, N.Y.).

Typically, stringent conditions are conditions under which saltconcentration is less than about 1.0M Na⁺. Typically, Na⁺ concentration(or other salt) is about 0.01 to 1.0M at pH of 7.0 to 8.3, and atemperature is at least about 30° C. for a short nucleotide (forexample, 10 to 50 nucleotides) and at least about 60° C. for a longnucleotide (for example, longer than 50 nucleotides). Stringentconditions also may be achieved by addition of a destabilizing agentsuch as formamide. Example of stringent conditions in the presentspecification include hybridization in buffer (50% formamide, 1M NaCl,1% SDS) (37° C.) and washing at 60° C. in 0.1×SSC.

As used herein, “polynucleotide hybridizing under stringent conditions”refers to well-known conditions commonly used in the art. By using apolynucleotide selected from polynucleotides of the present invention asa probe and by using colony hybridization technique, plaquehybridization technique or Southern blot hybridization technique or thelike, such a polynucleotide can be obtained. Specifically, the termmeans a polynucleotide which can be identified by hybridization at 65°C. in the presence of 0.7 to 1.0M NaCl using a filter immobilized withcolony- or plaque-derived DNA, followed by washing of the filter at 65°C. conditions using 0.1- to 2-fold concentration of SSC (Saline-sodiumcitrate) solution (composition of 1-fold concentration of SSC solutionis 150 mM sodium chloride and 15 mM sodium citrate). Hybridization canbe performed in accordance with a method described in experiment bookssuch as Molecular Cloning 2nd ed., Current Protocols in MolecularBiology, Supplement 1-38, DNA Cloning 1: Core Techniques, A PracticalApproach, Second Edition, Oxford University Press (1995). Here, from asequence hybridizing under stringent conditions, preferably, a sequenceincluding A bases alone or T bases alone is excluded. “Hybridizablepolynucleotide” refers to a polynucleotide capable of hybridizing toother polynucleotide under the aforementioned hybridization conditions.Examples of hybridizable polynucleotide specifically include apolynucleotide having at least 60% homology with a base sequence of DNAwhich encodes a polypeptide having an amino acid sequence specificallydescribed in the present invention, preferably a polynucleotide having80% or more homology or polynucleotide having 90% or more homology, andmore preferably, a polynucleotide having 95% or more homology.

Amino acids may be mentioned herein by either three-letter code thereofas generally known or one-letter code recommended by IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides may also be mentionedby one-letter code as generally recognized.

As used herein, “homology” of a gene refers to a degree of identity toeach other among two or more gene sequences. Thus, the higher thehomology of two genes is, the higher the identity or similarity of thesequences is. Whether or not two genes have homology can be investigatedby direct comparison of the sequences, or in a case of nucleic acid,hybridization under stringent conditions, or the like. In a case ofdirect comparison of two gene sequences, when DNA sequences havetypically at least 50% identity, preferably at least 70% identity, morepreferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity,between the gene sequences, the genes have homology.

Herein, comparison of similarity, identity and homology of amino acidsequence and base sequence are calculated using default parameters withBLAST, a tool for sequence analysis. Identity search can be performed byusing NCBI BLAST 2.2.9 (published May 12, 2004). Value of identitydescribed herein normally refers to a value in a case of alignment underdefault conditions using the BLAST. However, when a higher value isgiven by change of parameter, the highest value is employed as a valueof identity. When identity is evaluated in a plurality of regions, thehighest value thereof is employed as a value of identity.

As used herein, “corresponding” gene refers to a gene having orpredicted to have in a species an action similar to a given gene of aspecies which is the basis of comparison. When a plurality of geneshaving such action exist, “corresponding” gene refers to a gene havingevolutionarily the same origin. Thus, a gene corresponding to a gene(for example, EGFR) may be ortholog of the gene. Thus, a genecorresponding to a human gene can also be found in other animals (suchas mouse, rat, pig, rabbit, guinea pig, bovine and ovine). Such acorresponding gene can be identified using well-known techniques in theart. Accordingly, for example, a corresponding gene in an animal can befound by searching in sequence database of the animal (for example,mouse, rat, pig, rabbit, guinea pig, bovine, ovine or the like) using asequence of a gene which is the basis of the corresponding gene as aquery sequence.

As used herein, “fragment” refers to a polypeptide or polynucleotidehaving a sequence length up to 1 to n−1 with respect to the full-lengthpolypeptide or polynucleotide (length: n). A length of a fragment can beappropriately changed depending on the purpose. Examples of a lowerlimit of the length include 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40,50 and more amino acids. A length represented by an integer notspecifically listed here (for example, 11) may also be appropriate as alower limit. Furthermore, in a case of polynucleotide, examples of lowerlimit include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 andmore nucleotides, and a length represented by an integer notspecifically listed here (for example, 11) may also be appropriate as alower limit. Herein, a length of polypeptide and polynucleotide can berepresented by the number of amino acids and nucleic acids,respectively, as described above. The aforementioned numbers are notabsolute. It is intended that the aforementioned numbers as an upperlimit or lower limit also include numbers some more or less (or, forexample, 10% more or less) than the aforementioned numbers, as long asthe same function is possessed. In order to express such intention,herein, the numbers may be expressed with “about” attached before thenumbers. However, it should be understood that herein the presence orabsence of “about” does not influence on interpretation of the numericalvalue. Herein, a length of useful fragment may be determined dependingon whether or not at least one of functions of the full-length proteinwhich is the basis for the fragment is retained.

As used herein, “variant,” “modified sequence” or “analog” refers towhat includes partial change with respect to an original substance suchas polypeptide or polynucleotide, which preferably retains substantiallyat least one of functions of the original polypeptide or polynucleotide.Examples of such variant include substitution variant, addition variant,deletion variant, truncated variant, allelic mutant and the like. Allelerefers to one member of a pair of distinct genetic variants located atthe same gene locus. Therefore, “allelic mutant” refers to a variant ina relationship of allele of a certain gene. “Species homolog or homolog”refers to what has homology (preferably 60% or more homology, and morepreferably 80% or more, 85% or more, 90% or more, and 95% or morehomology) to a given gene in certain species at the amino acid ornucleotide level. A method for obtaining such a species homolog isapparent from the description of the specification.

In the specification, in order to produce a functionally equivalentpolypeptide, an amino acid addition, deletion, or modification can becarried out in addition to an amino acid substitution. An amino acidsubstitution refers to replacement of an amino acid of an originalpeptide with one or more (for example, 1 to 10, preferably 1 to 5, andmore preferably 1 to 3) amino acids. An amino acid addition refers toaddition of one or more (for example, 1 to 10, preferably 1 to 5, andmore preferably 1 to 3) amino acids to an original peptide. An aminoacid deletion refers to deletion of one or more (for example, 1 to 10,preferably 1 to 5, and more preferably 1 to 3) amino acids from anoriginal peptide. An amino acid modification includes, but is notlimited to, amidation, carboxylation, sulfation, halogenation,alkylation, phosphorylation, hydroxylation, acylation (for example,acetylation), and the like. An amino acid to be substituted or added maybe a naturally occurring amino acid, a non-naturally occurring aminoacid, or an amino acid analog. A naturally occurring amino acid ispreferable.

Such a nucleic acid can be obtained by well-known PCR technique, and canalso be synthesized chemically. For example, site specific mutagenesistechnique, hybridization technique or the like may be combined with sucha method.

As used herein, “substitution, addition and/or deletion” of apolypeptide or a polynucleotide refers to replacement, addition, orremoval of an amino acid or a substitute thereof, or a nucleotide or asubstitute thereof, in an original polypeptide or polynucleotide. Such asubstitution, addition and/or deletion technique is well known in theart, including, for example, site specific mutagenesis technique. Thesechanges in the nucleic acid molecule or polypeptide which is the basismay be caused at 5′ or 3′ terminus of the nucleic acid molecule, atamino terminal site or carboxyl terminal site of an amino acid sequencewhich indicates this polypeptide, or may be caused anywhere betweenthese terminal sites, and separately spread among residues in thesequence as the basis, as long as the desired function (for example,binding to TPR domain) is retained. Any number of substitution, additionor deletion is possible, as long as the number is one or more. Such anumber can be increased, as long as the desired function (for example,binding to TPR domain) is retained in a variant having suchsubstitution, addition or deletion. For example, the number may be oneor several, and may be preferably 20% or less, 15% or less, 10% or lessor 5% or less of a full length, or 150 or less, 100 or less, 50 or less,25 or less, or the like.

(Production and Analysis of Peptide)

The peptide of the present invention (for example, chimeric peptide) maybe obtained or produced by a method well known in the art (for example,chemical synthesis and general industrial technique discussed below).For example, a peptide corresponding to a part of a peptide including adesired region or domain or a peptide which mediates a desired activityin vitro may be synthesized by use of a peptide synthesizer. A peptidemay also be analyzed by hydrophilicity analysis which may be used foridentification of hydrophobic and hydrophilic regions of a peptide (see,for example, Hopp and Woods, 1981. Proc. Natl. Acad. Sci. USA.78:3824-3828), and thus is a help in designing a substance forexperimental manipulation (for example, binding test or antibodysynthesis). Secondary structure analysis may also be performed in orderto identify a region of a peptide which establishes a particularstructural motif (see, for example, Chou and Fasman, 1974, Biochem.13:222-223) Manipulation, translation, prediction of secondarystructure, hydrophilicity and hydrophobicity profiles, prediction andplotting of open reading frame and determination of sequence homologymay be achieved using a computer soft program available in the art.Examples of other method for structural analysis include X-ray crystalanalysis (see, for example, Engstrom, 1974. Biochem. Exp. Biol.11:7-13)); mass spectrometry and gas chromatography (see, for example,METHODS IN PROTEIN SCIENCE, 1997. J. Wiley and Sons, New York, N.Y.).Computer modeling (see, for example, Fletterick and Zoller, ed., 1986.Computer Graphics and Molecular Modeling: CURRENT COMMUNICATION INMOLECULAR BIOLOGY, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) may also be used.

The present invention further relates to a nucleic acid encoding apeptide of the present invention having an L-amino acid. An appropriatesource of a nucleic acid encoding the peptide of the present inventionincludes a human genome sequence. As other source, rat genome sequenceis included. Protein sequences are respectively available from GenBank,and the entirety thereof is incorporated herein as a reference. Anucleic acid encoding a peptide may be obtained by any method known inthe art (for example, by PCR amplification using a synthetic primerhybridizable to 3′- and 5′-terminuses of a sequence and/or by cloningfrom a genome library using an oligonucleotide sequence specific forcDNA or a predetermined gene sequence).

For expression of a recombinant of a peptide, a nucleic acid includingall or a part of the nucleic acid sequence which encodes the peptide maybe inserted in an appropriate expression vector (i.e., a vectorincluding elements necessary for transcription and translation of theinserted peptide coding sequence). In some embodiments, a regulatoryelement is heterologous (i.e. not native gene promoter). Or, necessarytranscription signal and translation signal may be provided by apromoter native for a gene and/or a region adjacent thereto. Varioushost vector systems may be used for expression of a sequence encoding apeptide. They include, but are not limited to: (i) mammalian cell systeminfected with vaccinia virus, adenovirus or the like; (ii) insect cellsystem infected with baculovirus or the like; (iii) yeast includingyeast vector; or (iv) bacteria transformed with bacteriophage DNA,plasmid DNA or cosmid DNA. Depending on a host cell system to be used,any one of a number of suitable transcription elements and translationelements may be used.

As a promoter/enhancer sequence in an expression vector, plant, animal,insect or mycotic regulatory sequence provided in the present inventionmay be used. For example, a promoter/enhancer element may be used fromyeast and other mycete (for example, GAL4 promoter, alcoholdehydrogenase promoter, phosphoglycerol kinase promoter, alkaliphosphatase promoter). Examples of expression vector or derivativethereof include human or animal virus (for example, vaccinia virus oradenovirus); insect virus (for example, baculovirus); yeast vector;bacteriophage vector (for example, λ phage); plasmid vector and cosmidvector.

As a host cell line, expression of a desired sequence inserted may beregulated, or an expressed peptide encoded by the sequence may bemodified, treated or selected by a particular desired means.Furthermore, expression from a particular promoter may be enhanced inthe presence of a particular inducer in a selected host cell line,thereby facilitating control of expression of a generally designedpeptide. Furthermore, a different host cell has a particularcharacterized mechanism for translation and post-translational processesand modification of the expressed peptide (for example, glycosylation,phosphorylation or the like). Thus, an appropriate cell line or hostcell system may be selected for guaranteeing that a desired modificationand process of a foreign peptide has been achieved. For example, peptideexpression in bacterial system may be used for producing anon-glycosylated core peptide. On the other hand, expression inmammalian cell guarantees “native” glycosylation of a heterologouspeptide.

Derivatives, fragments, homologs, analogs and mutants of a peptide, andnucleic acids encoding these peptides are included. Regarding nucleicacids, the derivatives, fragments and analogs provided herein aredefined as a nucleic acid sequence of at least six (contiguous), andhave a length sufficient for specific hybridization. Regarding aminoacids, the derivatives, fragments and analogs provided herein aredefined as an amino acid sequence of at least four (contiguous) and havea length sufficient for specific recognition.

In designing a variant, based on sequential information of otherreceptor and the like described in the following documents, a similarreceptor-binding peptide can be designed: for IL-13, Yuichiro Yoshida etal., Biochem Biophys Res Commun. 2007, vol. 358, No. 1, pp. 292-7; forIL-4, Thorsten Hage et al., Cell. 1999, vol. 97, No. 2, pp. 271-81; forneuropilin-1, Alexander Antipenko et al., Neuron. 2003, vol. 39, No. 4,pp. 589-98; for Transferrin R, Jae H. Lee et al., Eur. J. Biochem. 2001,vol. 268, pp. 2004-2012; for VEGFR1 (WHSDMEWWYLLG (SEQ ID NO: 31)), PingA N et al., 2004, Int. J, Cancer. Vol. 111, pp. 165-173; for HER-2,Valeria R. Fantin et al., Cancer Res. 2005, vol. 65, No. 15, pp.6891-6900, Stephanie C. Pero et al., Int J Cancer. 2004, vol. 111, pp.951-960, Beihai Jiang et al., J Biol Chem. 2006, vol. 280, No. 6, pp.4656-4662; for VEGFR1 (VEPNCDIHVMWEWECFERL-NH2 (SEQ ID NO: 32)),Kimberly J. Peterson et al., Analytical Biochemistry 2008, vol. 378, No.1, pp. 8-14; for VEGFR1 (GGNECDAIRMWEWECFERL (SEQ ID NO: 33)), BorlanPan et al., J. Mol. Biol. 2002, vol. 316, No. 3, pp. 769-87; forBuforin, Hyun Soo Lee et al., Cancer Lett. 2008, vol. 271, No. 1, pp.47-55; for FGFR (MQLPLAT (SEQ ID NO: 5)), Fukuto Maruta et al., CancerGene Therapy. 2002, vol. 9, pp. 543-552; for FGFR (AAVALLPAVLLALLAP (SEQID NO: 6)), Akiko Komi et al., Exp. Cell Res. 2003, Vol. 283, No. 1, pp.91-100; for NRP1/VEGFR2 (ATWLPPR(SEQ ID NO: 36)), Loraine Tirand et al.,J. Control Release. 2006, vol. 111, pp. 153-164; for EphB1 and EphB2,Mitchell Koolpe et al., J. Biol. Chem. 2005, vol. 280, No. 17, pp.17301-11; for IL11R, Amado J. Zurita et al., Cancer Res. 2004, vol. 64,pp. 435-439; for GRP78 (WDLAWMFRLPVG (SEQ ID NO: 39)), Marco A. Arap etal., Cancer Cell. 2004, vol. 6, pp. 275-284; for GRP78 (CTVALPGGYVRVC(SEQ ID NO: 40)), Ying Liu et al., Mol. Pharmaceutics. 2007, Vol. 4, No.3, pp. 435-447; for PSMA, Kaushal Rege et al., Cancer Res. 2007, vol.67, No. 13, pp. 6368-6375 (these documents are incorporated herein as areference). Such modification includes, but is not limited to,conservative substitution. Here, IL4-, IL13- and neuropilin-1-bindingsequence were designed based on results of three-dimensional structureanalysis. In the specification, for HER2, VEGFR and TfR, activity wasfound in those which used the binding sequence information per sedescribed in the aforementioned documents, as well as in EGFR.

Furthermore, regarding modification of a cell-permeable peptide,modification can be carried out with reference to conventional knowledgeand based on the description herein. For example, Daniele Derossi etal., THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 30, Issue of July26, pp. 18188-18193, 1996 provides knowledge related to a mechanism ofAntp and a variant thereof obtained by adding a partial mutation. Itdescribes a site important for cell permeation, and can be referenced inproduction of a variant or analog of the present invention. Thisdocument is incorporated herein as a reference in entirety thereof.

As other document, Genevie Ave Dom et al., Nucleic Acids Research, 2003,Vol. 31, No. 2 556-561; Wenyi Zhang and Steven O, Smith, Biochemistry2005, 44, 10110-10118; and ISABELLE LE Roux, et al., Proc. Natl. Acad.Sci. USA Vol. 90, pp. 9120-9124, October 1993 provide informationrelated to transmambrane mechanism and mutation. These documents canalso be referenced in production of a variant or analog of the presentinvention. These documents are incorporated herein as a reference inentirety thereof.

(Drug)

A compound of the present invention or a pharmaceutically acceptablesalt thereof can be administered alone, but is preferably providednormally as various pharmaceutical preparations. Furthermore, suchpharmaceutical preparations are used for animals and human.

It is preferred to use the administration route most effective intherapy. Examples of administration route include oral route orparenteral route such as intrarectal route, buccal route, subcutaneousroute, intramuscular route, intravenous route and the like. Examples ofadministration form include capsule, tablet, granule, powder, syrup,emulsion, suppository, injection and the like. Liquid preparationssuitable for oral administration, such as emulsion and syrup, can beproduced using water, saccharides such as sucrose, sorbitol, fruit sugarand the like; glycols such as polyethylene glycol, propylene glycol andthe like; oils such as sesame oil, olive oil, soybean oil and the like;a preservative such as p-hydroxybenzoic acid esters; flavors such asstrawberry flavor, peppermint and the like. Furthermore, capsules,tablets, powders, granules and the like can be produced using anexcipient such as lactose, glucose, sucrose, mannitol and the like; adisintegrator such as starch, soda alginate and the like; a lubricantsuch as magnesium stearate, talc and the like; a binding agent such aspolyvinyl alcohol, hydroxypropyl cellulose, gelatin and the like; adetergent such as fatty acid ester and the like; and a plasticizer suchas glycerin and the like.

Preparations suitable for parenteral administration are preferablycomposed of sterilized aqueous preparation containing an active compoundwhich is isotonic to the blood of an acceptor. For example, in a case ofinjection, a solution for injection is prepared using a carrier formedof salt solution, glucose solution or a mixture of salt water andglucose solution.

A topical preparation is prepared by dissolving or suspending an activecompound in one or more solvent (for example, mineral oil, petroleum,polyvalent alcohols, or other base used in topical pharmaceuticalpreparation. A preparation for intestinal administration is preparedusing a normal carrier, for example, cacao butter, hydrogenated fat,hydrogenated fatty carboxylic acid, and the like, and is provided as asuppository.

In the present invention, also in a parenteral preparation, one or moreauxiliary components selected from glycols, oils, flavors, preservatives(including antioxidant), excipients, disintegrators, lubricants, bindingagents, detergents, plasticizer and the like exemplified in relation toan oral preparation.

An effective dose and the number of administrations of the compound ofthe present invention or a pharmaceutically acceptable salt thereof varydepending on administration form, age and weight of a patient, natureand severity of a symptom to be treated, and the like. Normally, a doseis 0.01 to 1000 mg/person per day, preferably 5 to 500 mg/person.Regarding the number of administrations, it is preferred to administerone time per day or administer separately.

The present invention also relates to a system, device and kit forproducing a pharmaceutical composition of the present invention. It isunderstood that elements known in the art can be used as elements ofsuch a system, device and kit, which can be appropriately designed bythose skilled in the art.

The present invention also relates to a system, device and kit using acompound of the present invention, a pharmaceutically acceptable saltthereof, or a prodrug such as a hydrate thereof. It is understood thatelements known in the art can be used as elements of such a system,device and kit, which can be appropriately designed by those skilled inthe art.

(DDS)

As used herein, “delivery agent” or “delivery medium” refers to acarrier (vehicle) which mediates delivery of a substance of interest. Ifa substance to be delivered is a drug, it is referred to as “drugdelivery medium.” Drug Delivery System (DDS) may be classified toabsorption-controlling DDS, release-controlling DDS, and targeting DDS.An ideal DDS is a system which delivers “a necessary amount” of a drug“to a necessary site of a body” “for a necessary time.” Targeting DDS isclassified to passive targeting DDS and active targeting DDS. The formeris a method of controlling behavior in a body utilizing physicochemicalproperties such as a particle size and hydrophilicity of the carrier(drug carrier or drug vehicle) The latter is a method in which a specialmechanism is added to them to actively control directivity to a targetedtissue. For example, there is a method using a carrier conjugated withan antibody having a function of specific molecule recognition for atarget molecule of a particular cell composing the target tissue (forexample, TPR-binding peptide of the present invention), which may bealso referred to as “missile drug.”

As used herein, “drug delivery medium” refers to a vehicle fordelivering a desired drug.

As used herein, “a substance of interest” particularly refers to asubstance desired to be delivered into a cell.

As used herein, “liposome” normally refers to a closed vesicle composedof a lipid layer gathering in a membrane shape and a water layer inside.In addition to phospholipids typically used, it is also possible toincorporate cholesterol, glycolipids and the like. Since liposome is aclose vesicle containing water inside, it is also possible to retain awater-soluble drug or the like in the vesicle. Accordingly, such aliposome is used for delivering a drug or gene which cannot pass thecell membrane into a cell. Furthermore, due to good biocompatibility,liposome is significantly expected as a nano particle carrier materialfor DDS. In the present invention, in order to add a modification group,by optionally using a linker, a crosslinking agent or the like, liposomecan be possessed as a constitutional unit having a functional group togive an ester bond (for example, glycolipids, ganglioside, phosphatidylglycerol or the like) or a constitutional unit having a functional groupto give a peptide bond (for example, phosphatidyl ethanolamine).

Liposome can be prepared by any method known in the art. For example,among known methods, a method by cholic acid dialysis is included. Incholic acid dialysis, production is performed by a) preparation of mixedmicelle of lipids and detergent and b) dialysis of the mixed micelle.Next, regarding a sugar chain liposome used in the present invention, ina preferred embodiment, a protein is preferably used as a linker, andcoupling of glycoprotein in which sugar chain has been bound to aprotein to a liposome can be performed by the following two-stepreaction: a) periodate oxidation of ganglioside portion on liposomalmembrane and b) coupling of glycoprotein to oxidized liposome by areducing amination reaction. By such a method, it is possible to bind aglycoprotein including a desired sugar chain to a liposome, therebyobtaining various glycoprotein-liposome conjugates having a desiredsugar chain. For observing purity and stability of liposome, it is veryimportant to investigate particle size distribution. As a methodtherefor, gel permeation chromatography (GPC), scanning electronicmicroscope (SEM), dynamic light scattering (DLS) and the like can beused.

As used herein, “linker” refers to a molecule which mediates binding ofa surface-binding molecule (for example, TRP-binding peptide) and othermolecule (for example, liposome surface). In a sugar chain-modifiedliposome used in the present invention, a peptide may be bound to aliposome surface via a linker. A linker can be appropriately selected bythose skilled in the art, but is preferably biocompatible, and morepreferably, pharmaceutically acceptable. As used herein, “linkerprotein” refers to protein, peptide, amino acid polymer among linkermolecules.

As used herein, “linker (protein) group” is a name given when a linker(protein) has bound to other group. A linker (protein) group) refers tomonovalent or bivalent group, depending on the case. Examples thereofinclude mammal-derived protein group, human-derived protein group, humanserum protein group and serum albumin group. A linker (protein) group ispreferably derived from “human” because it is believed to have highbiocompatibility in administration to human. Furthermore, a proteinwithout immunogenicity is preferred.

As used herein, “cross linking group” refers to a group which formschemical bond between molecules of chain macromolecules like a bridge.Thus, this term partly overlaps with “linker” as a concept. Typically,this term refers to a group which acts between a macromolecule such aslipids, proteins, peptides, sugar chains and the like and other molecule(such as lipids, proteins, peptides and sugar chains) to form covalentbond linking a portion which lacked covalent bond in the molecule orbetween molecules. In the present specification, a crosslinking groupvaries depending on a target to be crosslinked, and examples thereofinclude, but are not limited to, aldehydes (such as glutaraldehyde),carbodiimides, imido esters and the like. When a substance containing anamino group is crosslinked, an aldehyde-containing group, for example,glutaraldehyde can be used.

As used herein, “biocompatibility” refers to a property of beingcompatible to an organism tissue or organ without causing toxicity,immune response, damage and the like. Examples of biocompatible bufferinclude, but are not limited to, phosphate buffered saline (PBS),saline, Tris buffer, carbonate buffer (CBS),Tris(hydroxymethyl)methylaminopropane sulfonate buffer (TAPS),2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonate (HEPES), otherGood's buffer (for example, 2-morpholinoethanesulfonic acid, monohydrate(MES), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-tris),N-(2-acetamide)iminodiacetic acid (ADA),1,3-bis[tris(hydroxymethyl)methylamino]propane (Bis-tris propane),piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES),N-(2-acetamide)-2-aminoethanesulfonic acid (ACES), coramine chloride,N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-morpholinopropanesulfonic acid (MOPS),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),N-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid (HEPPS),N-[tris(hydroxymethyl)methyl]glycine (Tricine), amino acetamide (glycineamide), N,N-bis(2-hydroxyethyl)glycine (Bicine),N-cyclohexyl-2-aminoethanesulfonic acid (CHES),N-cyclohexyl-3-aminopropanesulfonic acid (CAPS)) and the like.

As described above, the present invention provides a delivering agentfor delivering a substance of interest to a cancer cell, which containsa receptor-binding peptide such as EGFR domain-binding peptide. Asubstance of interest may or may not be conjugated with areceptor-binding peptide such as EGFR domain-binding peptide. Whenconjugated, the substance becomes a fusion substance, and in a case ofpeptide, such a peptide is referred to as chimeric peptide. A chimericpeptide of the present invention can be also regarded as thisembodiment. Regarding such a substance, a conjugate agent may be formedusing a medium (vehicle). As such a medium, liposome can be used, and asubstance of interest may be either outside of the liposome or includedinside. (Screening)

As used herein, “screening” refers to selecting a target such as anorganism or substance having a particular property of interest from apopulation including a mass by a particular manipulation/evaluationmethod. For screening, a specific moiety of the present invention can beused.

As used herein, for example, performing screening using immune responseis also referred to as “immunophenotyping”. In this case, a peptide ofthe present invention may be used for classification of cell lines andbiological samples. The present invention is useful as a cell-specificmarker, or more particularly, as a cell marker which is distinctivelyexpressed at various stages of differentiation and/or maturation of aparticular cell type. A monoclonal antibody directed to specific epitopeor a combination of epitope allows screening of cell population whichexpresses a marker. Various techniques may be used for screening a cellpopulation which expresses the marker, by using a monoclonal antibody.Examples of such a technique include magnetic separation using magneticbeads coated with antibody, “panning” using antibody attached to a solidmatrix (i.e. plate) and flow cytometry (see, for example, U.S. Pat. No.5,985,660 and Morrison et al., Cell, 96:737-49 (1999)).

For example, it can be utilized for screening a cell populationincluding undifferentiated cells (for example, embryonic stem cells,tissue stem cells and the like), such as a cell population which mayoccur cellular growth and/or differentiation as may be found in humanumbilical cord blood, or cell population in which modification treatmentto untreated state has been performed.

The references quoted herein, such as scientific articles, patents,patent applications or the like are incorporated herein as a referencein entirety thereof, to the same extent as respectively described in aspecific manner.

Hereinafter, the present invention will be described based on Examples.The Examples described below are provided only for the purpose ofillustration. Thus, the scope of the present invention is limitedneither by the aforementioned embodiments nor the Examples below but islimited only by the claims attached hereto.

EXAMPLES

Hereinafter, the present invention will be described in further detailby way of Examples, but the technical scope of the invention is notlimited by such Examples. Reagents used in the Examples described belowcan be obtained from Nakalai Tesque, Sigma-Aldrich, Wako Pure ChemicalIndustries, Ltd. or the like, unless particularly indicated. Animalexperiments were conducted based on the standard determined by KyotoUniversity and in accordance with the spirit of animal protection.

Example 1 EGFR-Targeted Chimeric Peptide

[Materials and Methods]

(Cell Lines)

Human breast cancer (BT-20 and T47D), lung cancer (H322 and H460),pancreatic cancer (SU.86.86), prostate cancer (LNCaP), brain tumor(U251), and lung fibroblast (MRC-5 and WI-38) cell lines were purchasedfrom the American Type Culture Collection (Manassas, Va.). Humanpancreatic cancer cell line (BxPC-3) and colon cancer cell line (HCT116and DLD-1) were purchased from the European Collection of Cell Cultures(ECACC; Salisbury, Wiltshire, UK). Human embryonic kidney cell line(HEK293) was purchased from RIKEN Cell Bank (Tsukuba, Japan). Cells werecultured in RPMI1640 (BT-20, T47D, H322, H460, SU.86.86, LNCaP, U251,BxPC-3, DLD-1 and SW837), MEM (MRC-5 and WI-38), McCoy's 5a (HCT116) orD-MEM (HEK293) containing 10 μl % FBS (BioWest, Miami, Fla.), 100 μg/mlpenicillin and 100 g/ml streptomycin (Nakalai Tesque, Kyoto, Japan).

(Peptides)

The following peptides were purchased from Invitrogen, Carlsbad, Calif.:

1. cancer cell membrane-lytic peptide: KLLLKLLKKLLKLLKKK (SEQ ID NO: 1;the underlined letters represent D-amino acids);

2. EGFR binding (EB)-cancer cell membrane-lytic chimeric peptide:YHWYGYTPQNVIGGGKLLLKLLKKLLKLLKKK (SEQ ID NO: 2);

3. original lytic peptide: LKLLKKLLKKLLKLL-NH₂ (SEQ ID NO: 41); and

4. EB-original lytic chimeric peptide:YHWYGYTPQNVIGGGLKLLKKLLKKLLKLL-NH₂ (SEQ ID NO: 42).

The peptides were chemically synthesized using solid phase chemistry,purified by high performance liquid chromatography until they becamehomogeneous (i.e. purity higher than 90%), and evaluated by massspectrometry. The peptides were dissolved in water, and buffered to pH7.4. The peptide solutions were newly prepared for each time just beforeuse so as to prevent reuse.

(Drugs)

Gefinitib and Erlotinib were purchased from Toronto Research Chemicals(Ontario, Canada). Anti-EGFR mouse monoclonal antibody (clone 225) andPD153035 were purchased from Calbiochem (La Jolla, Calif.).

(Preparation of Small Unilamilar Vesicles (SUVs))

Small unilamilar vesicles (SUVs) were prepared as described previously(Matsuzaki, K. & Horikiri, C. Interactions of Amiloid β-peptide (1-40)with Ganglioside-containing membranes. Biochemistry 38, 4137-4142(1999)). Briefly, a lipid film of the desired composition was dispersedin water or Tris buffer (10 mM Tris/150 mM NaCl/1 mM EDTA, pH7.4). Theresulting MLVs were subjected to five freeze-thaw cycles, and thensubjected to ultrasonic treatment in ice-cold water under a nitrogenatmosphere for 15 minutes using a probe-type sonicator (Tomy UD-201).Metal debris from the titanium tip of the probe was removed bycentrifuge. The lipid concentration was determined in triplicate byphosphorous analysis (Bartlett, G. R. Phosphorus assay in columnchromatography. J. Biol. Chem. 234, 466-468 (1959)).

(CD Spectra)

CD spectra were measured in Jasco J-820 using 1 mm path-length quartzcell to minimize the absorbance due to buffer components. For eachsample, an average of eight scannings was determined. The averaged blankspectra (small vesicle suspension or solvent) were subtracted. Thepeptide and lipid concentrations were 50 μM and 4 mM, respectively.

Visualization of membrane permeabilization (Imura Y., Choda, N., &Matsuzaki, K. Magainin 2 in action: distinct modes of membranepermeabilization in living bacterial and mammalian cells. Biophys. J.95, 5757-5765 (2008)). Calcein, a soluble fluorescence molecule, wasadded to MDA-MB-231 cell in a glass-bottomed dish at a finalconcentration of 2 μM. Small aliquots of labeled peptide,EB-Lytic-TAMRA-OH or Lytic-TAMRA-OH (Invitrogen) (15 μl) were directlyadded to the dish at a final concentration of 10 μM. Using OlympusFV1000 confocal laser scanning microscope (Olympus), confocal imageswere taken.

(Cell Viability Assay)

A total of 3×10³ cells per well were seeded in 96-well plates, culturedfor 24 hours in a medium containing 10% FBS, and incubated withincreasing concentrations of peptide in 100 μl for 48 to 72 hours at 37°C. Cell viability was measured with WST-8 solution (Cell Count ReagentSF; Nakalai Tesque).

(Immunofluorescence Staining)

EGFR expression by flow cytometry was determined by incubation of 1×10⁵cells with the human monoclonal antibody to EGFR conjugated with FITC(Santa Cruz). All stainings were performed at room temperature for 40minutes. The cell fluorescence was measured by flow cytometry(FACSCalibur, Becton Dickinson, San Jose, Calif.). The mean fluorescenceintensity (MFI) of EGFR-positive cells was determined using theCellQuest software (Becton Dickinson).

(Annexin V Assay and Caspase Assay)

BT-20 cells were treated for 2 hours at 37° C. with or without EB-lyticchimeric peptide at 5 μM. For determination of caspase activation orAnnexin V-positive expression, peptide-treated cultures weresimultaneously analyzed for caspase activity and propidium iodide (PI)staining using a carboxyfluorescein FLICA caspase-3&7 assay(Immunochemistry Technologies, Bloomington, Minn.), or, alternatively,for Annexin V labeling and PI staining by multiparametric flowcytometry. Furthermore, in accordance with the manufacturer'sinstructions, using a confocal laser scanning microscope, terminaldeoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay wasconducted by MEBSTAIN Apoptosis Kit Direct (MBL).

(Biomolecular Interaction)

Surface plasmon resonance (SPR) experiments were performed with aBIACORE biosensor system 3000 (BIACORE Inc., Uppsala, Sweden). About5000 RU of streptavidin (Sigma) was immobilized on the surface of CM5sensor chips via N-hydroxysuccinimide andN-ethyl-N′-(dimethylaminopropyl) carbodiimide activation chemistry, andthen 2000-3000 RU of peptide conjugated with biotin were injected overthe streptavidin-immobilized sensor chip. As a control of nonspecificbinding, the unreacted carboxymethyl groups of a sensor chip withoutimmobilized streptavidin were blocked with ethanolamine. As an analyte,cell surface proteins which were prepared using the Mem-PER eukaryoticmembrane protein extraction reagent kit (Pierce) were injected over theflow-cell in the flow rate of 201/min. at 25° C. In order to preventnonspecific binding during the assay, HBS buffer (0.01 M HEPES, 0.15 MNaCl, 0.005% Tween 20, 3 mM EDTA [pH 7.4]) was used as running buffer.Interaction analysis of recombinant human EGF receptor (rhEGFR) withEB-lytic chimeric peptide was carried out as following: about 5000RU ofrhEGFR (SEQ ID NO: 3) was immobilized on the sensor chip of CM5 asdescribed above, and then several concentrations of peptides wereinjected over this sensor chip. All protein concentrations used in theseexperiments were determined by the Bradford method (Bradford M M. Arapid and sensitive method for the quantitation of microgram quantitiesof protein utilizing the principle of protein-dye binding. Anal Biochem1976; 72:248-54). Data analysis was performed using BIA evaluationver.3.2 software (BIACORE).

(Colony-Forming Assay)

The in vitro cytotoxic activity of the lytic peptide or the chimericpeptide against H322 cells was determined by a colony-forming assay. Thecells were seeded in 6 cm dishes with 3 ml of RPMI 1640 containing 10%FBS and were allowed to attach. The number of cells/dishes was set sothat >100 colonies were obtained in the control group. Twenty four hoursafter seeding the cells, the cells were exposed to differentconcentrations of lytic peptide or EB-lytic chimeric peptide (0 to 22.5μM) and cultured for 10 days. The dishes were washed with phosphatebuffer, and stained with crystal violet (0.25% in 25% alcohol). Thepercentage of colony survival was determined from the number of coloniesformed in the control and treated groups.

[Results]

(Designing of a Novel Chimeric Peptide Based on a Secondary Structure)

Previously, Papo and Shai have reported a new lytic peptide composed ofa 15-amino acid diastereomeric sequence containing D- and L-leucine andlysine (Papo, N. & Shai, Y. New Lytic peptides based on theD,L-amphipathic helix motif preferentially kill tumor cells compared tonormal cells. Biochemistry 42, 9346-9354 (2003)). In this study, theinventors designed a novel lytic peptide which is suitable forcombination with EGFR-binding (EB) peptide, based on amphipathicity in asecondary structure. As shown in FIG. 1D, the location of cluster ofpositively charged amino acids (Lys) in the newly designed lytic peptidewas still retained after the combination with EB peptide in comparisonwith that of EB-lytic peptide (FIG. 1D a and b). CD spectra analysisdemonstrated that EB-lytic peptide weakly bound to small unilamellarvesicles (SUVs) composed of phosphatidylcholine (PC), which is thedominant lipid species on the surface of normal cell membranes, and wasnot well structured with the PC liposome, while this EB-lytic peptidewas capable of binding to SUVs containing phosphatidylserine (PS), whichis exposed specifically on cancer cell membranes, and conformed to apartial helical structure as characterized by double minima at 209-210and 222 nm (FIG. 1D (d)). On the other hand, EB-original lytic peptidewas capable of strongly binding to both PC and PC/PS liposomes, andconformed to helical structures (FIG. 1D(c)). These results indicatethat the chimeric peptide newly designed in this study has a selectivityto PS-containing membranes and conforms to a helical structure which issupposed to be essential for making a pore on the cell surface (Papo, N.& Shai, Y. New Lytic peptides based on the D,L-amphipathic helix motifpreferentially kill tumor cells compared to normal cells. Biochemistry42, 9346-9354 (2003)).

(EGFR Targeting Enhances the Cytotoxic Action of the Lytic Peptide inEGFR-Expressing Cancer Cells)

The cytotoxicity of the lytic peptide was compared with theEGFR-targeted peptidetoxin, an EGFR-binding binding-lytic chimericpeptide, against seven EGFR expressing cancer cell lines. As shown inFIG. 1A, treatment with the lytic peptide or chimeric peptide resultedin a concentration-dependent cytotoxicity in all cancer cell linestested. The chimeric peptide demonstrated considerable enhancement incytotoxic activity to cancer cells, when compared with the lytic peptidealone. A 15 to 20 μM concentration of the chimeric peptide wassufficient to induce more than 80% of cell death in all the cell lines.In contrast, the same concentration of the lytic peptide alone could notinduce sufficient cell killing of cancer cells. As shown in Table 1, thelytic peptide upon EGFR targeting enhanced the IC₅₀ (the peptideconcentration inducing 50% inhibition of control cell growth) for cancercells by 1.6- to 3.1-fold, suggesting that the EGFR targeting enhancedsusceptibility of cancer cells to the EB-lytic chimeric peptide incomparison with the lytic peptide alone. The inventors then assessed thecytotoxicity of the EB-lytic chimeric peptide and the lytic peptidealone in three normal cell lines. As shown in FIG. 1B, three normal celllines including MRC-5, WI-38 and HEK293 were less susceptible to thelytic peptide, demonstrating less cytotoxicity compared to cancer celllines. The IC₅₀ of the chimeric peptide in normal cell lines was 3.6- to8-fold higher for normal cells than for cancer cells (Table 1). TheEB-lytic chimeric peptide also enhanced the cytotoxicity to normalcells, which is similar to the phenomena in cancer cells. These findingssuggest that the lytic peptide has stronger cytotoxic activity in cancercells than in normal cells and that the EGFR-targeted peptidetoxin hassuperior cytotoxic activity to cancer cells with high EGFR expression.Interestingly, EB peptide combined with the original lytic peptide didnot show enhancement of cytotoxic activity, and moreover, theEB-original lytic peptide killed even normal cell line (MRC-5) at lowerconcentration of the peptide, suggesting that the original lytic peptideis not suitable for chimerization with EB peptide (FIG. 1C).

(Treatment with the EB-Lytic Chimeric Peptide Results in SufficientCytotoxicity to Cancer Cells Resistant to Tyrosine Kinase Inhibitor(TKI))

In cancer cells with or without k-ras mutation, cytotoxic activity ofthe EB-lytic peptide and TKI were compared.

(Evaluation of Sensitivity of k-Ras Mutated Cancer Cells to TKI)

Sensitivity of various cancer cells to erlotinib or anti-EGFR antibodyin the presence and absence of k-ras mutation was studied.

A total of 3×10³ cells per well of k-ras wild-type (WT) cancer celllines (H322 and BT-20) and k-ras mutated cancer cell lines (MDA-MB-231,HCT116, SW837 and DLD-1) were seeded in 96-well plates, and incubatedfor 24 hours in a medium containing 10% FBS. These cells were culturedwith various concentrations of erlotinib (0 to 80 μM) or anti-EGFRantibody (right: 0 to 20 μg/ml) for 72 hours, and cytotoxic activity wasassessed using WST-8 reagent (Cell Count Reagent SF; Nakalai Tesque).

The k-ras WT cancer cell lines (H322 and BT-20) were sensitive toerlotinib and anti-EGFR antibody, but the k-ras mutated cancer celllines (MDA-MB-231, HCT116, SW837 and DLD-1) were resistant to botherlotinib and anti-EGFR antibody (FIG. 1E).

(Cytotoxicity of TKI or EB-Lytic in k-Ras Wild-Type Cells)

Cytotoxicity of TKI or EB-lytic chimeric peptide to cancer cells withoutk-ras mutation and normal cells was studied.

A total of 3×10³ cells per well of three k-ras wild-type (WT) cancercell lines (H322, BT-20 and BxPC-3) and lung normal cell line (MRC-5)were seeded in 96-well plates, and incubated for 24 hours in a mediumcontaining 10% FBS. These cells were cultured with variousconcentrations of TKI (erlotinib, gefitinib and PD153035; 0 to 20 μM) orthe EB-lytic chimeric peptide (0 to 20 μM) for 72 hours, and cytotoxicactivity was assessed using WST-8 reagent (Cell Count Reagent SF;Nakalai Tesque).

The treatment with the three TKIs (erlotinib, gefinitib and PD153035)resulted in concentration-dependent growth inhibition in the k-ras WTcancer cell lines (H322, BT-20 and BxPC-3), but the cytotoxic activitywas insufficient. On the other hand, treatment with EB-lytic peptideexhibited sufficient cytotoxic activity to these cancer cell lines, butdid not exhibit cytotoxic activity to the lung normal cell line MRC-5(FIG. 1F).

(Cytotoxic Activity of the EB-Lytic to TKI-Resistant Cancer Cell Linewith k-Ras Mutation)

Cytotoxicity of the EB-lytic chimeric peptide to TKI-resistant cancercells with k-ras mutation was studied.

A total of 3×10³ cells per well of four k-ras mutated cancer cell lines(MDA-MB-231, HCT116, SW837 and DLD-1) were seeded in 96-well plates, andincubated for 24 hours in a medium containing 10% FBS. These cells werecultured with various concentrations of TKI (erlotinib, gefitinib andPD153035; 0 to 20 μM) or the EB-lytic chimeric peptide (0 to 20 μM) for72 hours, and cytotoxic activity was assessed using WST-8 reagent.

The k-ras mutated cancer cell lines (MDA-MB-231, HCT116, SW837 andDLD-1) were resistant to erlotinib, gefitinib and PD153035, but thetreatment with EB-lytic chimeric peptide exhibited sufficient cytotoxicactivity to the TKI-resistant cancer cell lines with k-ras mutations(FIG. 1G).

Thus, the EB-lytic chimeric peptide revealed to exhibit cytotoxicactivity specific for k-ras mutated cancer cell lines.

(The Degree of Enhancement of Cytotoxic Activity Induced by the EB-LyticChimeric Peptide Depends on Expression Levels of EGFR on the CellSurface)

The inventors next examined whether or not the increase in cytotoxicityof the EB-lytic chimeric peptide correlated with the expression levelsof EGFR on the cell surface. The expression levels of EGFR for sevencancer cell lines and three normal cell lines were assessed by flowcytometry using an FITC-conjugated anti-EGFR polyclonal antibody. Asshown in FIG. 2A and Table 1, the expression levels of EGFR did notcorrelate with IC₅₀ of the EB-lytic chimeric peptide or lytic peptide(r=−0.37 for the chimeric peptide (FIG. 2A, left) and r=−0.14 for thelytic peptide (FIG. 2A, center)). However, the expression levels of EGFRsufficiently correlated with IC₅₀ ratio of the lytic peptide to theEB-lytic chimeric peptide, suggesting that the degree of enhancement ofcytotoxic activity by the EB-lytic chimeric peptide as compared withthat by the lytic peptide alone depends on the expression levels of EGFRon the cell surface (r=0.89; FIG. 2A, right).

To further confirm the specificity of EB-lytic chimeric peptide to EGFR,anti-EGFR polyclonal antibody (Ab) or recombinant human EGF were addedto the BxPC-3 culture, one hour prior to the exposure to the EB-lyticchimeric peptide to assess the cytotoxic activity to cells. As shown inFIG. 2B, both EGF protein and EGFR-Ab were capable of blocking thecytotoxicity of EB-lytic chimeric peptide, demonstrating 27% inhibitionby EGF ligand and 23% inhibition by EGFR-Ab. These results suggest thatthe binding of EB-lytic chimeric peptide to cells depends on theexpression levels of EGFR on the cell surface.

(Interaction Profile for Binding of the EB-Lytic Chimeric Peptide toEGFR Protein and Cell Surface Membrane Proteins)

To understand the binding property of peptides to EGFR, EGFR protein wasimmobilized on sensor chips and interaction profile with EB-lyticchimeric peptide or lytic peptide alone was analyzed using BIACORE. Asshown in FIG. 3A, the resonance signal intensity increased according tothe concentrations of EB-lytic chimeric peptide, indicating that theamount of EB-lytic chimeric peptide bound to EGFR protein isproportional to the increase in the concentrations of EB-lytic chimericpeptide. In contrast, the resonance signal intensity by lytic peptidealone minimally increased according to the concentrations. The K_(D)value for EB-lytic chimeric peptide binding to EGFR protein was 2.6×10⁻⁵(M) Next, to understand the binding property of peptides to cells,either EB-lytic chimeric peptide or lytic peptide alone was immobilizedon sensor chips, and interaction profile with cell surface membraneproteins extracted from H322, BT-20, or MRC-5 was analyzed usingBIACORE. As shown in FIGS. 3B and 3C, the resonance signal intensityincreased according to the concentrations of cell membrane proteins,indicating that the amount of cell membrane proteins bound to thepeptide is proportional to the increase in the concentrations of cellmembrane proteins. Interaction of cell membrane proteins to lyticpeptide alone demonstrated similar binding constants in each cell linewith increased level of the peptide. On the other hand, bindingconstants of EB-lytic chimeric peptide to H322 or BT-20 cancer cellmembrane proteins were 4.2-fold (H322) or 4.4-fold (BT-20) stronger thanlytic peptide alone (FIGS. 3B, 3C and 3D). In contrast, binding constantto MRC-5 normal cell membrane proteins did not vary significantly ascompared to lytic peptide alone (FIG. 3D). These results were consistentwith the data obtained from WST assays (FIG. 1), indicating that thecytotoxic activity of EB-lytic chimeric peptide correlated well with theaffinity to the cell membranes.

(EB-Lytic Chimeric Peptide Induces Rapid Killing of Cancer Cells)

To assess the appropriate time duration of EB-chimeric peptide to killcancer cells, H322 or BT-20 cells were treated with either EB-chimericpeptide or lytic peptide alone for 10 minutes, 30 minutes, one hour, or48 hours. As shown in FIG. 4, treatment of H322 or BT-20 cells withlytic peptide alone resulted in loss of viability in the time-dependentmanner. In contrast, a mere 10-minute exposure of H322 or BT-20 cells toEB-chimeric peptide (10 μM) sufficiently killed cancer cells, and morethan 70% of cell-killing effect was exhibited. Confocal microscopeanalysis also demonstrated that this chimeric peptide penetrates thecell membrane to make the pore on the cancer cell surface. The influx ofcalcein-labeled medium to cytosol of cancer cells was observed within 20minutes (FIG. 4B). However, this rapid penetration was not observed inthe case of lytic peptide alone (FIG. 4C). These results suggest thatEB-chimeric peptide kills cancer cells quite rapidly as compared tolytic peptide alone. In vitro colony-forming assay also demonstratedthat this chimeric peptide inhibits the cell growth of H322 cancer cellsin concentration-dependent manner (FIG. 4D).

(EB-Chimeric Peptide Induces Caspase Activation and Annexin V-PositiveExpression in Cancer Cells)

To investigate the cell death mechanism of action caused by EB-chimericpeptide, an Annexin V assay and a caspase assay using multiparametricflow cytometry analysis were performed. As shown in FIG. 5A twohour-exposure of EB-lytic chimeric peptide (5 μM) to BT-20 breast cancercells induced caspase activation and Annexin V-positive expression. Fromthese results, EB-lytic chimeric peptide disintegrated the plasmamembrane of cancer cells, and thus it appears that the chimeric peptideinduced cell death of the cancer cells by an apoptotic mechanism.

(In Vitro Inhibition of H322 Cancer Cell Growth)

To further confirm the cytotoxic activity of EB-lytic chimeric peptideto H322 cancer cells, colony-forming assay was performed (Kawakami K,Kawakami M, Leland P, Puri R K. Internalization property ofinterleukin-4 receptor alpha chain increases cytotoxic effect ofinterleukin-4 receptor-targeted cytotoxin in cancer cells. Clin CancerRes 2002; 8:258-66; Kawakami K, Joshi B H, Puri R K. Sensitization ofcancer cells to interleukin 13-pseudomonas exotoxin-induced cell deathby gene transfer of interleukin 13 receptor alpha chain. Hum Gene Ther2000; 11:1829-35). As shown in FIG. 6, although H322 cells weresensitive to lytic peptide alone (IC₅₀, 14.2 μM), the cells showed atleast two times higher sensitivity to the chimeric peptide (IC₅₀<7.5μM). The IC₅₀ values of two peptides by colony-forming assay correlatewell with the IC₅₀ values determined by WST assay.

TABLE 1 Cytotoxicity of peptides to various cell lines and EGFRexpression IC₅₀ (μM) IC₅₀ Relative MFI * Lytic peptide EB-lytic Ratio(anti-EGFR alone peptide Lytic/ antibody, %) Cell lines mean ± SD mean ±SD EB-lytic mean ± SD Cancer cells H322 21 ± 3.2 6.8 ± 0.5  3.1 90 ± 23 BT-20 20 ± 2.9 6.5 ± 0.7  3.1 100 U251 20 ± 2.5 12 ± 2.0 1.6 39 ± 5.6H460 20 ± 1.6 8.9 ± 1.6  2.3 13 ± 3.6 BxPC-3 32 ± 1.6 12 ± 0.9 2.7 64 ±17  SU.86.86 28 ± 0.5 12 ± 2.3 2.3 43 ± 4.6 LNCaP 16 ± 2.5 10 ± 1.3 1.620 ± 7.0 Normal cells WI-38 100 ± 3.1  46 ± 2.7 2.2 45 ± 4.6 MRC-5 110 ±8.1  49 ± 5.8 2.3 47 ± 13  HEK293 58 ± 0.3 44 ± 2.8 1.3  0 * Therelative MFI (mean fluorescence intensity) is the extent of binding ofthe FITC-conjugated anti-EGFR polyclonal antibody to cells, where themean MFI values for BT-20 and HEK293 cells are set at 100% and 0%,respectively.[Discussion]

In this study, the inventors linked two functional domains of aminoacids to produce a novel chimeric peptide termed “peptidetoxin” of thepresent invention, which was designed as a bifunctional peptide bindingto EGFR to lyse the plasma membrane for the targeting ofEGFR-overexpressing cancer cells. The inventors found that the EB-lyticchimeric peptide kills cancer cells more rapidly and efficiently whencompared with lytic peptide alone. On the other hand, the inventorsfound that normal cells are not very sensitive to both peptides.

The inventors propose a mechanism of action of EB-lytic chimeric peptidein cancer killing as follows. First, EB portion of the chimeric peptidebinds EGFR on the cell surface, followed by binding of the lytic moietyof the chimeric peptide and disintegration of the cell membrane morerapidly than free lytic peptide. EB peptide bound specifically andefficiently to EGFR with a dissociation constant of 22 nM (Li Z, Zhao R,Wu X, et al. Identification and characterization of a novel peptideligand of epidermal growth factor receptor for targeted delivery oftherapeutics. FASEB J 2005; 19:1978-85). Therefore, it was suggestedthat the affinity of EB-EGFR interaction is greater than the affinity ofthe lytic peptide and cell membrane.

Similar to the lytic peptide, mitochondriotoxic and proapoptotic peptidehas polycationic sequence (KLAKLAK)₂ (SEQ ID NO: 4), invades intoendocytic compartment by a peptide having membrane-permeable sequencewithout disintegration of the cell membrane, and induces mitochondrialdamage (Ellerby H M, Arap W, Ellerby L M, et al. Anti-cancer activity oftargeted pro-apoptotic peptides. Nat Med 1999; 5:1032-8). A cellmembrane-lytic peptide invades in the cell after disintegration of thecell membrane, induces mitochondrial damage and activation of caspase,and triggers apoptosis. On the other hand, Pseudomonas exotoxin-basedimmunotoxins (interleukin-13-Pseudomonas exotoxin (IL13-PE38QQR) (SEQ IDNO: 6)) induce apoptosis partially, and mere 10-30% of head and neckcancer cells undergo apoptotic cell death (Kawakami M, Kawakami K, PuriR K. Apoptotic pathways of cell death induced by an interleukin-13receptor-targeted recombinant cytotoxin in head and neck cancer cells.Cancer Immunol Immunother 2002; 50:691-700). Cancer cells treated withthis hybrid peptide are Annexin V- and caspase 3,7-positive when assayedby flow cytometry. Furthermore, this peptide also induced rapid cancercell death. Thus, this targeted chimeric peptide has an advantage ofinducing cancer cell death rapidly as compared to bacterial toxin-basedimmunotoxin, and may be capable of inducing bystander action or naturalimmunity at the treatment site in vivo.

Generally, peptides are relatively easily inactivated by serumcomponents in human body (Papo N, Braunstein A, Eshhar Z, Shai Y.Suppression of human prostate tumor growth in mice by a cytoLyticD-,L-amino acid peptide:membrane lysis, increased necrosis, andinhibition of prostate-specific antigen secretion. Cancer Res 2004;64:5779-86). It has been shown that diastereomeric peptides arerelatively free from inactivation in serum, and lytic diastereomericpeptide administrated either intratumorally or intravenously reduces thetumor growth of animal models of human prostate cancer without rapiddegradation of the peptide in blood (Papo N, Braunstein A, Eshhar Z,Shai Y. Suppression of human prostate tumor growth in mice by acytoLytic D-, L-amino acidpeptide:membrane lysis, increased necrosis,and inhibition of prostate-specific antigen secretion. Cancer Res 2004;64:5779-86). Other type of peptide-based drug reported recently, whichbinds to Hsp90 in the cancer cell to destabilize its client proteins,also inhibits human breast cancer cell growth in mice with intravenousinjection in mice at the concentration of 50 mg/kg (Plescia, J. et al.,Rational design of shepherdin, a novel anticancer agent. Cancer Cell 7,457-468 (2005)). In this study, it was found that the intravenousadministration of EB-lytic peptide newly designed by the inventorsreduced the tumor growth at a lower concentration as compared to otherpeptide drug candidates, suggesting the high potential of this novelchimeric peptide as a novel and useful tool for cancer therapy. AlthoughEB-lytic chimeric peptide may be resistant to the inactivation in bloodflow, as a method for increasing more the efficacy of the peptide in invivo use, it is believed that use in combination with other material ortopical administration will be effective. For example, it has been shownthat atelocollagen, calf dermis type I collagen highly purified bypepsin treatment, is a fascinating drug delivery system (DDS) forprotein drug and siRNA (Fujioka K, Maeda M, Hojo T, Sano A. Proteinrelease from collagen matrices. Adv. Drug Deliv Rev 1998; 31:247-66;Takeshita F, Minakuchi Y, Nagahara S, et al. Efficient delivery of smallinterfering RNA to bone-metastatic tumors by using atelocollagen invivo. Proc Natl Acad Sci USA 2005; 102:12177-82). It has been reportedthat protein drugs administered topically together with atelocollagenare continuously released from the injected site over a long period oftime (Fujioka K, Maeda M, Hojo T, Sano A. Protein release from collagenmatrices. Adv Drug Deliv Rev 1998; 31:247-66). Thus, it is believed thatatelocollagen is sufficiently worthy of tests in combination with thepeptidetoxin of the inventors in in vivo model of human cancers.Currently, these possibilities are under investigation in the laboratoryof the inventors.

Immunotoxin is composed of a protein toxin having killing moiety and atargeting moiety linked thereto for selectivity for cancer cells, suchas a ligand or an antibody. Immunotoxins can be classified into twocategories which are chemical conjugates as first-generationimmunotoxins and recombinant protein as second-generation immunotoxins(Reiter Y, Pastan I. Recombinant Fv immunotoxins and Fv fragments asnovel agents for cancer therapy and diagnosis. Trens Biotechnol 1998;16:513-520). The conventional immunotoxins usually show hurdles inclinical use (for example, immunogenicity, undesirable toxicity,difficulty in production, limited half-life and production ofneutralizing antibody in a body) (Kreitman R J. Immunotoxins forTargeted Cancer Therapy. AAPS J 2006; 8:E532-51; Li Z, Yu T, Zhao P, MaJ. Immunotoxins and Cancer Therapy. Cell Mol Immunol 2005; 2:106-112;Posey J A, Khazaeli M B, Bookman M A, et al., A phase I trial of thesingle-chain immunotoxin SGN-10 (BR96sFv-PE40) in patients with advancedsolid tumors. Clin Cancer Res 2002; 8:3092-3029). However, becausepeptides can be synthesized chemically, production of peptides can beperformed with affordable cost as compared to protein drugs. Inaddition, various combinations of candidate peptides for targeting andtoxic moieties can be generally tested easily in preclinical settings.For example, a toxic moiety having tumoricidal activity, such asmitochondriotoxin (Ellerby H M, Arap W, Ellerby L M, et al. Anti-canceractivity of targeted pro-apoptotic peptides. Nat Med 1999; 5:1032-8) orantibiotics-like derivative peptides (Kim S, Kim S S, Bang Y J, Kim S J,Lee B J. In vitro activities of native and designed peptide antibioticsagainst drug sensitive and resistant tumor cell lines. Peptides 2003;24:945-953), can be utilized. As a targeting moiety, interleukin-11(Zurita A J, Troncoso P, Cardo-Vila M, Logothetis C J, Pasqualini R,Arap W. Combinatorial screenings in patients: the Interleukin-11receptor α as a candidate target in the progression of human prostatecancer. Cancer Res 2004; 64:435-439) and prostate-specific membraneantigen (PSMA; Rege K, Patel S J, Megeed Z, Yarmush M L. Amphipathicpeptide-based fusion peptides and immunoconjugates for the targetedablation of prostate cancer cells. Cancer Res 2007; 67:6368-6375), inaddition to EGFR, can be targeted. However, an explosive portion peptideneeds stronger cell-killing activity like plant or bacterial toxin, andmust be widely exploited together with a warhead peptide for targetingon cancer cells.

In conclusion, peptidetoxin provided by the present invention, thattargets unique protein on the cancer cells is a new possible tool forthe anticancer target therapy. The inventors propose the concept of thischimeric peptide, peptidetoxin, as a concept of immunotoxin of nextgeneration. The research and development of peptidetoxins will enable infuture an individualized treatment of cancer according to the individualprofile, i.e. treatment with a chimeric peptide which is targetedspecifically for resected tumor from patients. Ultimately, this strategymay be useful for the treatment of not only cancer but also otherdiseases.

Example 2 Exhaustive Analysis of EGF-Receptor Binding Peptides

In the present Example, experiments for determining whether or not it ispossible to use EGF receptor-binding peptide analogs (having the aminoacid sequence X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂ (SEQ ID NO: 8), wherein:

X₁ is Y, or an amino acid similar thereto which is S, H or F;

X₂ is H, or an amino acid similar thereto which is R or K;

X₃ is W, or an amino acid similar thereto which is Y, F or H;

X₄ is Y, or an amino acid similar thereto which is S, H or F;

X₅ is G, or an amino acid similar thereto which is A, V, I or L;

X₆ is Y, or an amino acid similar thereto which is S, H or F;

X₇ is T, or an amino acid similar thereto which is S, H or F;

X₈ is P, or an amino acid similar thereto which is hydroxyl proline;

X₉ is Q, or an amino acid similar thereto which is N;

X₁₀ is N, or an amino acid similar thereto which is S, H or F;

X₁₁ is V, or an amino acid similar thereto which is G, A, L or I; and

X₁₂ is I, or an amino acid similar thereto which is G, A, V or L.

Except for difference in peptide sequence, all protocols is inaccordance with Example 1.

(Protocol)

Among the aforementioned mutated peptides, first, paying attention tothe second H which has charge, K- and R-mutants were chemicallysynthesized. As described in Example 1, using BIACORE, binding affinitywith recombinant human EGFR was evaluated by comparison with that ofwild-type EGFR-binding peptide. Only for what had higher effect in theresults, chimeric peptide with the cancer cell membrane-lytic sequencewas synthesized, and cell-killing effect to human lung cancer cell lineH322 was compared between wile-type EGFR-binding chimeric peptide andthe cancer cell membrane-lytic alone.

(Results)

The results are shown in FIG. 7. In comparison with the wild-typepeptide, for K-substituted peptide, some enhancement of biosensorresponse was observed, and for R-substituted peptide, response of twofold of wild-type was observed (FIG. 7A). The cell-killing effect toH322 was enhanced more for the R-substituted chimeric peptide (blackcircle) than for wild-type EGFR-binding chimeric peptide (triangle)(FIG. 7B).

Example 3 Combination of Cancer Cell Membrane-Lytic Sequence (OnlyL-Amino Acid) and IL-4 Receptor (IL4R)-Targeted Sequence as a CancerCell-Targeted Sequence

In the present Example, it was investigated whether or not the sameeffect as in Example 1, such as enhancement of cell-killing effect, wasobserved using a chimeric peptide formed by combining the cancer cellmembrane-lytic sequence of KLLLKLLKKLLKLLKKK (all amino acids areL-amino acids; LyticL; SEQ ID NO: 27) and IL4R-targeted sequence. Thechimeric peptide sequence is as follows:

(SEQ ID NO: 18) IL4-LyticL: KQLIRFLKRLDRNGGGKLLLKLLKKLLKLLKKK.(Protocol)

Based on the results of binding three-dimensional structure analysis ofIL4R and IL-4, partial peptide sequence which is important for binding(KQLIRFLKRLDRN (SEQ ID NO: 26)) was designed and chemically synthesized.By BIACORE analysis, binding affinity with human recombinant IL4Rprotein was assessed. The aforementioned chimeric peptide wassynthesized, and in vitro cell-killing effect to human breast cancercell line MDA-MB-231 and human pancreatic cancer cell line BxPC-3 wasassessed.

(Results)

The results are shown in FIG. 8. For IL4R-binding peptide sequence(KQLIRFLKRLDRN (SEQ ID NO: 26)), binding affinity with human recombinantIL4R protein was observed by BIACORE analysis (FIG. 8A). ForIL4R-targeted cancer cell membrane-lytic chimeric peptide (IL4-LyticL),in both of the two cancer cell lines, cell-killing effect was enhancedas compared to the cancer cell membrane-lytic peptide alone (LyticL)(FIG. 8B). It was demonstrated that as a result of chimerization,contact for 10 minutes sufficiently induces the cancer cells (BxPC-3) tocell death (FIG. 8C).

Example 4 Combination of Cancer Cell Membrane-Lytic Sequence (OnlyL-Amino Acid) and IL-13 Receptor (IL13R)-Targeted Sequence as a CancerCell-Targeted Sequence

In the present Example, it was investigated whether or not the sameeffect as in Example 1, such as enhancement of cell-killing effect, wasobserved using a chimeric peptide formed by combining the cancer cellmembrane-lytic sequence of KLLLKLLKKLLKLLKKK (all amino acids areL-amino acids; LyticL; SEQ ID NO: 27) and IL13R-targeted sequence. Thechimeric peptide sequence is as follows:

IL13-LyticL: (SEQ ID NO: 19) KDLLLHLKKLFREGQFNGGGKLLLKLLKKLLKLLKKK.(Protocol)

Based on the results of binding three-dimensional structure analysis ofIL13R and IL-13, partial peptide sequence which is important for binding(KDLLLHLKKLFREGQFN (SEQ ID NO: 28)) was designed and chemicallysynthesized. By BIACORE analysis, binding affinity with humanrecombinant IL13R protein was assessed. The aforementioned chimericpeptide was synthesized, and in vitro cell-killing effect to human braintumor cell line U251 and human head and neck cancer cell line HN-12 wasassessed.

(Results)

The results are shown in FIG. 9. For IL13R-binding peptide sequence(KDLLLHLKKLFREGQFN (SEQ ID NO: 28)), binding affinity with humanrecombinant IL13R protein was observed by BIACORE analysis (FIG. 9A).For IL13R-targeted cancer cell membrane-lytic chimeric peptide(IL13-LyticL), in both of the two cancer cell lines, cell-killing effectwas enhanced as compared to the cancer cell membrane-lytic peptide alone(LyticL) (FIG. 9B). It was demonstrated that as a result ofchimerization, contact for 10 minutes sufficiently induces the cancercells (U251) to cell death (FIG. 9C).

Example 5 Combination of Cancer Cell Membrane-Lytic Sequence (OnlyL-Amino Acid) and Neuropilin-1 (NRP1)-Targeted Sequence as a CancerCell-Targeted Sequence

In the present Example, it was investigated whether or not the sameeffect as in Example 1, such as enhancement of cell-killing effect, wasobserved using a chimeric peptide formed by combining the cancer cellmembrane-lytic sequence of KLLLKLLKKKKLLKLLKKK (all amino acids areL-amino acids; LyticL; SEQ ID NO: 27) and NRP1-targeted sequence. Thechimeric peptide sequence is as follows:

Sema3A-LyticL: (SEQ ID NO: 20) NYQWVPYQGRVPYPRGGGKLLLKLLKKLLKLLKKK.(Protocol)

Based on the results of binding three-dimensional structure analysis ofNRP1 and a Sema3A, a ligand thereof, partial peptide sequence which isimportant for binding (NYQWVPYQGRVPYPR (SEQ ID NO: 29)) was designed andchemically synthesized. By BIACORE analysis, binding affinity with humanrecombinant NRP1 protein was assessed. The aforementioned chimericpeptide was synthesized, and in vitro cell-killing effect to humanpancreatic cancer cell line SU86.86 and human breast cancer cell lineSKBR3 was assessed.

(Results)

The results are shown in FIG. 10. For NRP1-binding peptide sequence(NYQWVPYQGRVPYPR (SEQ ID NO: 29)), binding affinity with humanrecombinant NRP1 protein was observed by BIACORE analysis (FIG. 10A).For NRP1-targeted cancer cell membrane-lytic chimeric peptide(Sema3A-LyticL), in both of the two cancer cell lines, cell-killingeffect was enhanced as compared to the cancer cell membrane-lyticpeptide alone (LyticL) (FIG. 10B).

Example 6 Combination of Cell Membrane-Lytic and Nucleic Acid-BindingSequence and EGFR-Targeted Sequence as a Cancer Cell-Targeted Sequence

In the present Example, it was investigated whether or not the sameeffect as in Example 1, such as enhancement of cell-killing effect, wasobserved using a chimeric peptide formed by combining cellmembrane-lytic and nucleic acid-binding sequence (RLLRRLLRRLLRK (SEQ IDNO: 13); hereinafter, abbreviated as buf) and EGFR-targeted sequence.The chimeric peptide sequence is as follows:

EGFbuf: (SEQ ID NO: 21) YHWYGYTPQNVIGGGGGRLLRRLLRRLLRK.(Protocol)

The aforementioned chimeric peptide (EGFbuf) and explosive portionpeptide alone (buf) were synthesized, and in vitro cell-killing effectto human lung cancer cell line H323, human prostate cancer cell lineDU145 and human lung normal cell line MRC-5 was assessed.

(Results)

The results are shown in FIG. 11. For EGFR-targeted chimeric peptide(EGFbuf), in both of the two cancer cell lines, cell-killing effect wasenhanced as compared to the cancer cell membrane-lytic peptide alone(buf) (FIG. 11A) Furthermore, when cell-killing effects of EGFbuf tolung cancer cell line H322 and lung normal cell line MRC-5 werecompared, cell-killing sensitiveness to cancer cells was high (FIG.11B).

Example 7 Combination of Cancer Cell Membrane-Lytic Sequence (L-,D-MixedAmino Acid Composition) and Three Binding Sequences to a Receptor withHigh Expression in Cancer Cells

In the present Example, it was investigated whether or not the sameeffect as in Example 1, such as enhancement of cell-killing effect, wasobserved using a chimeric peptide formed by combining cellmembrane-lytic sequence (KLLLKLLKKLLKLLKKK (underlined letters representD-amino acids, and the others are L-amino acids; SEQ ID NO: 1)) andthree binding sequences to a receptor with high expression in cancercells (her2, VEGF receptor, Transferrin receptor). The chimeric peptidesequences are as follows (underlined letters represent D-amino acids,and the others are L-amino acids):

(SEQ ID NO: 15) HER2-Lytic: YCDGFYACYMDVGGGKLLLKLLKKLLKLLKKK(SEQ ID NO: 16) VEGFR-Lytic: WHSDMEWWYLLGGGGKLLLKLLKKLLKLLKKK(SEQ ID NO: 17) TfR-Lytic: THRPPMWSPVWPGGGKLLLKLLKKLLKLLKKK.(Protocol)

By searching documents, the aforementioned three receptor-bindingpeptide sequences were found, and chimeric peptides with a cancer cellmembrane-lytic sequence (KLLLKLLKKLLKLLKKK; the underlined lettersrepresent D-amino acids; hereinafter, abbreviated as Lytic; SEQ IDNO: 1) were designed and chemically synthesized. The aforementionedchimeric peptides were synthesized, and in vitro cell-killing effect tohuman lung cancer cell line H322 and human lung normal cell line MRC-5was assessed.

(Results)

The results are shown in FIG. 12. For any of the three anticancertargeted cell membrane-lytic chimeric peptides, in human lung cancercell line H322, the cell-killing effect was enhanced as compared toLytic peptide alone (FIG. 12A) Furthermore, in vitro cell-killing effectto human lung normal cell line MRC-5 was milder as compared to that ofthe two cancer cell lines (FIG. 12B).

Example 8 Tests of Other Receptor Peptides with High Expression inCancer Cells

In the present Example, it is investigated whether or not other receptorpeptides with high expression in cancer cells can be used.

The peptides to be used are as follows. Except for difference in peptidesequence, all protocols are in accordance with Example 1.

(Cancer Cell Growth System Receptor)

Fibroblast growth factor receptor (FGFR): MQLPLAT (SEQ ID NO: 5) orAAVALLPAVLLALLAP (SEQ ID NO: 6)

(Angiogenesis System Receptor)

Human epidermal growth factor receptor type 2 (HER2): LLGPYELWELSH (SEQID NO: 52), ALVRYKDPLFVWGFL (SEQ ID NO: 53), KCCYSL (SEQ ID NO: 54),WTGWCLNPEESTWGFCTGSF (SEQ ID NO: 55) or DTDMCWWWSREFGWECAGAG (SEQ ID NO:56)

Neuropilin 1 (NRP1)/vascular endothelial growth factor receptor 2(VEGFR2): ATWLPPR (SEQ ID NO: 36)

Vascular endothelial growth factor receptor 1 (VEGFR1/Flt1):VEPNCDIHVMWEWECFERL-NH2 (SEQ ID NO: 32) or GGNECDAIRMWEWECFERL (SEQ IDNO: 33)

Ephrin B1 (EphB1): EWLS (SEQ ID NO: 37)

Ephrin B2 (EphB2): SNEW (SEQ ID NO: 38)

(Cytokine/Chemokine Receptors)

interleukin-11 receptor (IL11R): CGRRAGGSC (cyclic) (SEQ ID NO: 22)

(Other Receptors with High Expression in Cancer Cells)

glucose regulatory protein 78 (GRP78): WDLAWMFRLPVG (SEQ ID NO: 39),CTVALPGGYVRVC (cyclic) (SEQ ID NO: 40) or YPHIDSLGHWRR (SEQ ID NO: 58)

(Cancer Antigen Surface Protein)

prostate-specific membrane antigen (PSMA): CQKHHNYLC (SEQ ID NO: 35)

(Results)

Similar to the case of using EGFR, by BIACORE analysis, binding affinityspecific for each receptor can be confirmed, and it can be expected thatchimeric peptide of each binding sequence and a cancer cellmembrane-lytic sequence has enhanced cell-killing effect as compared tocancer cell membrane-lytic peptide alone.

Example 9 Therapy Using Anticancer Targeted Chimeric Peptide(Peptidetoxin)

Using the chimeric peptide produced in Example 1, therapeutic effect wasconfirmed. An anticancer targeted chimeric peptide (peptidetoxin) wasdesigned, which is formed by binding a warhead portion peptide whichbinds to a molecule such as a receptor with high expression in a cancercell and an explosive portion peptide which exhibits strong cell-killingeffect to the cancer cell. Antitumor effect in cancer-bearing animalmodels was studied.

(Protocol)

To 5-weeks-old female nude mice balb/c-nu/nu, human pancreatic cancercell line BxPC-3 (5×10⁶ cells/150 μl phosphate buffer) was injectedsubcutaneously. From day 5 after the transplantation, the EB-lyticchimeric peptide was intratumorally administered three times per weekfor three weeks at 0 mg/kg, 0.3 mg/kg, 1 mg/kg (50 μl phosphatebuffer/mouse). The tumor diameters were measured over time using anelectronic caliper, and the tumor volume (mm³) was calculated as longerdiameter×shorter diameter×shorter diameter×0.5.

(Results)

The results are shown in FIG. 13.

In the group administered with phosphate buffer, increase of the tumorwas observed over time, but in the group administered with the EB-lyticchimeric peptide, both in the group administered with 0.3 mg/kg and 1mg/kg, dilatation of the tumors was inhibited significantly. The dose of0.3 mg/kg is a very small amount, and sufficient antitumor effect isexpected also in human. This result reveals that such a peptide has aconsiderably strong effect as compared with conventional arts, althoughpeptides have been believed to be easily decomposed in a body and tohave a weak effect. Stabilization by peptide chemical modification orthe like or combination with DDS or the like further increases stabilityin the body, and enhancement of the drug effect can also be expected.

Considering the results of the present Example, theoretical explanationthat the results are data which demonstrate that the chimeric peptide isactual anticancer agent for human. That is, using the data shown in FIG.13, it is understood by those skilled in the art that the data can be“regarded equivalent” to data in human. Since the cancer cell linestransplanted were derived from human and many anticancer agentsclinically used underwent similar research process, this is regarded asa theory established in the art. Thus, from the results of the presentExample, it is understood that the chimeric peptide of the presentinvention can be affirmed as an “anticancer agent” for human.

Example 10 Antitumor Effect by Intravenous Administration of EB-Lytic

In the same manner as described in the previous section, therapeuticeffect was confirmed using the chimeric peptide produced in Example 1.In the present Example, antitumor effect by systemic administration wasexamined.

(Protocol)

To 5-week-old female nude mice balb/c-nu/nu, human pancreatic cancercell line BxPC-3 (5×10⁶ cells/1501 phosphate buffer) was injectedsubcutaneously. From day 5 after the transplantation, the EB-lytic(EB-Lytic) chimeric peptide was intravenously administered three timesper week for three weeks at 0 (control), 1 and 5 mg/kg, or as anexperimental system separate from such, at 0, 2, 5 and 10 mg/kg (50 μlphosphate buffer/mouse). The tumor diameters were measured over timeusing an electronic caliper, and the tumor volume (mm³) was calculatedas longer diameter×shorter diameter×shorter diameter×0.5.

(Results)

The results are shown in FIG. 14 and upper portion of FIG. 15A. Asapparent from the above description, FIGS. 14 and 15A respectively showresults of independent experimental systems.

For the group administered with phosphate buffer, increase of tumor wasobserved over time, but for the group administered with EB-lyticchimeric peptide, both 1 and 5 mg/kg, dilatation of tumor wassignificantly inhibited. The dose of 1 mg/kg is a very small amount insystemic administration, and a sufficient antitumor effect is expectedalso in humans. Furthermore, tumor volumes at day 55 in the groupsadministered with 2, 5 and 10 mg/kg were, as compared to the tumorvolume of the control group treated with saline (1310 mm³), 34% (440mm³, P<0.05), 30% (399 mm³, P<0.05) and 19% (244 mm³, P<0.01),respectively. Such results revealed that the peptide is very stronglyeffective as compared to conventional arts, although so far a peptidehas been immediately decomposed in a body and had a weak effect and ithas been believed systemic administration of a peptide is verydifficult. By stabilization by peptide chemical modification or thelike, combination with DDS or the like, and the like, stability in bodyis further increased, and enhancement of drug effect can also beexpected.

Example 11 Therapeutic Method

In the present Example, application to actual therapy is investigated.

Here, stabilization of kinetics in a body, together with discussion ofstabilization of kinetics in body, release control and the like usingDDS (Drug Delivery System), antitumor effect in cancer-bearing animalmodels is studied.

(DDS)

The EB-lytic chimeric peptide was mixed with atelocollagen, and usinggelled preparation for topical administration, inhibition ofdecomposition and release-controlling effect are studied. Furthermore,for systemic administration, formulation for exhibiting an effect ofinhibiting peptide decomposition without gelling is studied. For suchstudy, free profile analysis from atelocollagen mixture of the peptidein vitro is performed and the optimal percentage of atelocollagencontent is determined.

(Antitumor Effect in Cancer-Bearing Animal Models)

A gelled mixture for topical administration (EB-lytic chimeric peptideand atelocollagen) is intratumorally administered to nude mousecancer-bearing models to assess antitumor effect, in the same manner asin Example 9. Furthermore, the mixture for systemic administration isintravenously administered to assess antitumor effect in the samemanner. For both administration methods, tests for setting usage anddose are conducted.

By conducting such experiments, it can be expected that intratumoraladministration of the gelled mixture formulation of EB-lytic chimericpeptide and atelocollagen results in sufficient antitumor effect due toinhibition of peptide decomposition and release-controlling effect, evenif the number of administrations is reduced. For intravenousadministration of a non-gelled mixture for systemic administration,higher antitumor effect as compared to peptide alone is expected.

Example 12 In Vivo Test for Setting Dose

Using the chimeric peptide produced in Example 1, dose setting isstudied.

(Protocol)

To 5-weeks-old female nude mice balb/c-nu/nu, human pancreatic cancercell line BxPC-3 (5×10⁶ cells/1501 phosphate buffer) was injectedsubcutaneously. From day 5 after the transplantation, the EB-lyticchimeric peptide is intravenously administered three times per week forthree weeks at 0 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg or 5 mg/kg (50 μlphosphate buffer/mouse). The tumor diameters are measured over timeusing an electronic caliper, and the tumor volume (mm³) is calculated aslonger diameter×shorter diameter×shorter diameter×0.5.

Example 13 In Vivo Test for Setting Usage

Using the chimeric peptide produced in Example 1, usage setting isstudied.

(Protocol)

To 5-weeks-old female nude mice balb/c-nu/nu, human pancreatic cancercell line BxPC-3 (5×10⁶ cells/150 μl phosphate buffer) is injectedsubcutaneously. From day 5 after the transplantation, the EB-lyticchimeric peptide is intravenously administered three times per week forthree weeks or five times per week for one week at 1 mg/kg (50 μlphosphate buffer/mouse). The tumor diameters are measured over timeusing an electronic caliper, and the tumor volume (mm³) is calculated aslonger diameter×shorter diameter×shorter diameter×0.5.

Example 14 Tests in Other Cancer Cells

Using the chimeric peptide produced in Example 1, antitumor effect inother type of cancer was studied.

(Protocol)

To 5-weeks-old female nude mice balb/c-nu/nu, human breast cancer cellline MDA-MB-231 or human prostate cancer cell line DU145 (5×10⁶cells/150 μl phosphate buffer) was injected subcutaneously. From day 5after the transplantation, the EB-lytic chimeric peptide isintravenously administered three times per week for three weeks at 0.5mg/kg (50 μl phosphate buffer/mouse). The tumor diameters are measuredover time using an electronic caliper, and the tumor volume (mm³) iscalculated as longer diameter×shorter diameter×shorter diameter×0.5.

(Breast Cancer Cell Line MDA-MB-231)

To assess antitumor effect of EB-lytic chimeric peptide in human cancerxenograft model, in vivo antitumor activity of the EB-lytic chimericpeptide in tumor xenograft was studied. Breast cancer cell lineMDA-MB-231 (5×10⁶ cells/150 μl phosphate buffer) was subcutaneouslyinjected to the flank region of 7- to 9-weeks-old female nude micebalb/c-nu/nu (body weight: 17-21 g). At the time point when the tumorreached to the volume of 20 to 60 mm³, the animals were randomly dividedinto three groups, which were intravenously administered (50 μlinjection) with saline (control) or EB-lytic peptide (2 mg/kg or 5mg/kg) three times per week for three weeks (nine administrations intotal) The tumor diameters were measured over time using an electroniccaliper, and the tumor volume (mm³) was calculated as longerdiameter×shorter diameter×shorter diameter×0.5. At the time oftermination of the treatment, the mice were killed and the tumors wereextracted. After staining with hematoxyline, histological test wasconducted using optical microscope. All values are represented asmean±SD, and statistical analysis was performed by one-way ANOVA andDunnet test. Differences were considered to be significant at P<0.05.

The control group exhibited gradual tumor growth to reach 1885 mm³ atday 48. On the other hand, administration of the EB-lytic peptide (2mg/kg, 5 mg/kg or 10 mg/kg, intravenous administration of three timesper week) significantly inhibited tumor growth (FIGS. 15A (lowerportion) and 15B). Day 48, mean tumor volume was 933 mm³ in the groupadministered with 2 mg/kg, and 419 mm³ in the group administered with 5mg/kg (P<0.01 as compared with the mice of the control group). As shownin FIG. 15C, the number of tumor cells drastically reduced in the micetreated with the EB-lytic peptide (FIG. 15C, right panel). These resultsindicate that the newly designed EB-lytic hybrid peptide successfullyinduces tumor death.

Example 15 EB(H2R)-Lytic Chimeric Peptide

In the present Example, activity of EB(H2R)-Lytic chimeric peptide wasinvestigated.

For wild-type EB-Lytic (YHWYGYTPQNVIGGGKLLLKLLKKLLKLLKKK; SEQ ID NO: 2;underlines represent D-amino acids) and mutated EB (H2R)-Lytic(YRWYGYTPQNVIGGGKLLLKLLKKLLKLLKKK; SEQ ID NO: 43; underlined lettersrepresent D-amino acids), comparison of binding affinity was performedby BIACORE system. The results are shown in FIG. 16A. Furthermore, usingCD spectrum, secondary structure analysis was performed for EB-lyticpeptide and EB(H2R)-lytic peptide. The results are shown in FIG. 16B.The peptide concentrations were 50 μM. Except for differences in peptidesequence, the protocol is in accordance with Example 1.

(Cell Viability Assay)

Using lytic peptide, wild-type EB-lytic peptide and EB(H2R)-lyticpeptide, comparison of cytotoxicity to BT20 cells was performed. A totalof 3×10³ cells per well were seeded in 96-well plates, cultured for 24hours in a medium containing 10% FBS, and incubated with increasingconcentrations of peptide in 1001 for 48 to 72 hours at 37° C. Cellviability was measured with WST-8 solution (Cell Count Reagent SF;Nakalai Tesque). The results are shown in FIG. 16C.

(EB(H2R)-Lytic Enhances Cytotoxic Activity to Cancer Cells)

Cancer cell lines H322, BT-20, U251, BxPC-3, SU86.86 and LNCaP werecultured with various concentrations (0 to 20 μM) of EB-lytic chimericpeptide or EB(H2R)-lytic chimeric peptide for 72 hours, and cytotoxicactivity was assessed using WST-8 reagent. The results are shown in FIG.17A. Furthermore, normal cell lines MRC-5 and HEK293 were cultured withvarious concentrations (0 to 20 μM) of the aforementioned peptides for72 hours, cytotoxic activity was assessed, and cytotoxicity assay wasconducted as described above. The results are shown in FIG. 17B. Theresults indicate that the newly designed lytic peptide is suitable forchimeric peptide for enhancing cytotoxic activity to cancer cells.

(EB(H2R)-Lytic Induces Rapid Killing of Cancer Cells)

H322 cells were treated with EB-lytic chimeric (white columns) orEB(H2R)-lytic chimeric peptide (black columns) for two minutes, fiveminutes, 10 minutes, 30 minutes, one hour, two hours or 24 hours, andthen the medium containing peptides was replaced with a fresh medium.The cells were further cultured for 24 hours. The cells were analyzedfor cell viability using WST-8. The results are shown in FIG. 18. Theresults indicate that the EB-lytic chimeric peptide permeates the plasmamembrane and induces rapid killing of cancer cells.

The results are summarized in Table 2.

TABLE 2 Cytotoxicity of peptides to various cell lines and EGFRexpression. IC₅₀ (μM) IC₅₀ Ratio Relative MFI * EB-lytic EB(H2R)-lyticEB-lytic peptide/ (anti-EGFR peptide peptide EB(H2R)-lytic antibody, %)Cell lines mean mean peptide mean ± SD Cancer cells H322 6.8 3.2 2.1 90± 23  BT-20 6.5 1.9 3.4 100 U251 12 8.4 1.4 39 ± 5.6 BxPC-3 12 6.6 1.864 ± 17  SU.86.86 12 7.4 1.6 43 ± 4.6 LNCaP 10 4.8 2.1 20 ± 7.0 Normalcells MRC-5 49 >20 47 ± 13  HEK293 44 >20  0 * The relative MFI (meanfluorescence intensity) is the extent of binding of the FITC-conjugatedanti-EGFR polyclonal antibody to cells, where the mean MFI values forBT-20 and HEK293 cells are set at 100% and 0%, respectively.(EB(H2R)-Lytic Disintegrates Cancer Cell Membrane More Efficiently thanEB-Lytic)

Ability of disintegrating cancer cell membrane of EB(H2R)-Lytic andEB-Lytic was studied.

Calcein, a soluble fluorescence molecule, was added to H322 lung cancercells in a glass-bottomed dish at a final concentration of 2 μM. Smallaliquots of Lytic peptide alone, EB-Lytic or EB(H2R)-Lytic (15 μl) weredirectly added to the dish at a final concentration of 10 μM. At 0minute, two minutes, five minutes, 10 minutes and 20 minutes afteraddition of peptide, using Olympus FV1000 confocal laser scanningmicroscope (Olympus), confocal images were taken. The images are shownin FIG. 19A.

From the images shown in FIG. 19A, it is seen that for Lytic peptidealone (upper) the number of cells which became green (cells with themembrane disintegrated) did not change even after lapse of time whilefor EB-Lytic peptide (center) and EB(H2R)-Lytic peptide (lower) thenumber of cells which became green increased over time and that thenumber increases in shorter time for EB(H2R)-Lytic peptide than forEB-Lytic peptide.

FIG. 19B is a graph showing percentage of influx of medium to the cellsalong lapse of time as calculated based on the results of FIG. 19A. Thisfigure also indicates that the percentage of influx to the cells wasmore efficient for EB(H2R)-Lytic peptide than EB-Lytic peptide.

It is recognized that it revealed as a result of cell membranepermeation by calcein solution that EB(H2R)-Lytic is more efficient thanEB-Lytic. Regarding FIGS. 19A and B, as negative control, themicroscopic data for Lytic peptide alone in FIG. 19A can be employed.That is, it is difficult for this Lytic peptide alone to attainmembrane-lytic ability. FIG. 19B is a graph of percentage of influx ofcalcein-containing medium. Thus, it is clearly seen that the membrane isclearly penetrated earlier for EB (2R)-Lytic.

(EB (H2R)-Lytic Induces Cell Death by an Apoptotic Mechanism in CancerCells More Strongly than EB-Lytic)

The ability of inducing Annexin V-positive expression in cancer cells byEB(H2R)-Lytic and EB-Lytic was studied.

In accordance with the method of Example 1, Annexin V assay and caspaseassay were performed. Specifically, BT20 cells were treated for twohours at 37° C. with or without EB-Lytic or EB(H2R)-Lytic chimericpeptide at 5 μM. For determination of caspase activation or AnnexinV-positive expression, peptide-treated cultures were simultaneouslyanalyzed for caspase activity and propidium iodide (PI) staining using acarboxyfluorescein FLICA caspase-3&7 assay (ImmunochemistryTechnologies, Bloomington, Minn.), or, alternatively, for Annexin Vlabeling and PI staining by multiparametric flow cytometry. The resultsare shown in FIG. 20A (Annexin V) and FIG. 20B (caspase activity)

Regarding FIG. 20A, it is recognized that EB(H2R)-lytic is superior toEB-lytic in inducing Annexin V-positive expression. In BT-20 cellstreated with respective peptides, the ratio of right half panel showingAnnexin V-positive cells, and EB(H2R)-lytic induced Annexin V-positiveexpression more strongly.

Furthermore, regarding FIG. 20B, it is recognized that induction ofcaspase activity of EB(H2R)-lytic is superior to EB-lytic. In BT-20cells treated with the respective peptides, the ratio of the right halfpanel showing caspase 3,7-activated cells, and EB(H2R)-lytic inducedcaspase 3,7 activation more strongly.

From a viewpoint of an excellent anticancer agent, it is believed thatcausing suicide to avoid scattering therearound is much moreadvantageous than killing when side effects and other influences in vivoare taken into consideration. Regarding the hybrid peptide of thepresent invention, the killing mechanism hardly gives influence such asdisorder to cells around a tumor in that the possibility of action isspecific to cancer cells and the induction of apoptosis was suggested.Thus, the hybrid peptide of the present invention can be regardedexcellent as a pharmaceutical. Furthermore, this evaluation system wasemployed due to its importance in accurate calculation of AnnexinV-positive expression and caspase 3,7 activation which are importantindicators of apoptosis.

Furthermore, in the present Example, it revealed that, when the peptideof the present invention was administered, live cells significantlydecreased. Thus, it can also be proof that the peptide of the presentinvention selectively kills more cancer cells, regardless of whether ornot it mediates an apoptotic mechanism.

Moreover, it can be recognized that the present Example demonstratedwhether EB-lytic and EB (H2R)-lytic are excellent in cell-killing effectin view of degree and selectivity which cannot be achieved withconventional anticancer agents.

(Antitumor Effect of EB-Lytic Chimeric Peptide and EB (H2R)-LyticChimeric Peptide in Mouse Models Bearing Human Breast Cancer Cell LineMDA-MB-231)

In mouse models bearing human breast cancer cell line MDA-MB-231,antitumor effect of EB-Lytic chimeric peptide and EB(H2R)-Lytic chimericpeptide was studied.

MDA-MB-231 breast cancer cells were subcutaneously transplanted toathymic nude mice. From day 5 after the transplantation, the animalswere divided into three groups (n=6/group), and saline (control),EB-lytic peptide (1 mg/kg) or EB (H2R)-Lytic chimeric peptide (1 mg/kg)was intravenously injected. Results of measurement of tumor diametersover time are shown in FIG. 21.

As apparent from the figure, it is seen that the chimeric peptide inwhich the second H of the EB peptide has been changed from H into R(EB(H2R)-Lytic) has higher antitumor effect in vivo as compared toEB-Lytic chimeric peptide (EB-Lytic).

The results of FIG. 4 are results of comparison at a concentration ofpeptide of 1 mg/kg. Based on the results, it should be noted that EB(H2R)-lytic can attain sufficient antitumor effect even in systemicadministration at 1 mg/kg. As apparent from other results, it isunderstood that administration of 5 mg/kg results in anticancer actionof a considerable therapy level. Regarding administration of 1 mg/kg,EB(H2R)-lytic still attained sufficient effect. This should be regardedas just a surprising numerical value in view of the conventional stateof art.

Specifically, EB(H2R)-Lytic exhibited sufficient antitumor effect evenat a concentration of 1 mg/kg, and it is seen that EB(H2R)-Lytic is avery promising anticancer therapeutic drug.

Example 16 TfR-Lytic Chimeric Peptide

In the present Example, activity of TfR-Lytic chimeric peptide wasinvestigated.

(EfR-Lytic Enhances Cytotoxic Activity to Cancer Cells)

Using TfR-Lytic chimeric peptide (THRPPMWSPVWPGGGKLLLKLLKKLLKLLKKK; SEQID NO: 17; underlined letters represent D-amino acids), cytotoxicactivity to cancer cells was studied.

A total of 3×10³ cells per well of cancer cell lines T47D, MDA-MB-231and SKBR-3 were cultured with various concentrations (0 to 30 μM) ofTfR-lytic chimeric peptide or lytic peptide for 72 hours, and cytotoxicactivity was assessed using WST-8 reagent. The results are shown in FIG.22A. Furthermore, normal cell lines MRC-5, PE and HC were cultured withvarious concentrations (0 to 100 μM) of the aforementioned peptides for72 hours, and cytotoxic activity was assessed. The absolute valueobtained from untreated cells was defined as 100%. The results are shownin FIG. 22B. Furthermore, regarding enhancement of cytotoxicity byaddition of TfR-lytic chimeric peptide, IC₅₀ of TfR-lytic chimericpeptide and IC₅₀ of lytic peptide, or IC₅₀ ratio of lyticpeptide/TfR-lytic chimeric peptide were studied. Correlation betweenIC₅₀ values was observed. These results indicate that TfR-lytic peptideis suitable for chimeric peptide for enhancing cytotoxic activity tocancer cells. The results are summarized in Table 3.

TABLE 3 Cytotoxicity of peptides to various cell lines and TfRexpression. IC₅₀ (μM) Lytic TfR-lytic IC₅₀ ratio peptide alone peptideLytic/ MFI * Cell lines Mean Mean TfR-lytic Mean Cancer cells T47D 18.24.8 3.8 425.7 MDA-MB-231 29.5 6.7 4.4 428.7 SK-BR-3 26.3 5.3 5.0 533.6LNCaP 17.0 6.2 2.7 284.0 U251 19.0 6.8 2.8 188.9 SN-19 25.3 8.2 3.1184.1 COLO587 13.9 6.9 2.0 179.2 Normal cells MRC-5 >20 >20 — 64.6 HCcell >20 >20 — 89.6 (hepatocyte cell) PE cell >20 >20 — 41.2 (pancreaticepidermal) * The MFI (mean fluorescence intensity) is the extent ofbinding of the PE-conjugated anti-Transferrin receptor monoclonalantibody to cells.(TfR-Lytic Induces Rapid Killing Specific for TfR-Expressing CancerCells)

It is indicated that TfR-lytic peptide permeates plasma membrane torapidly kill T47D cancer cells. T47D cells were treated with TfR-lyticchimeric peptide (black columns) or lytic peptide (white columns) forvarious periods of time (1 to 180 minutes), and then the mediumcontaining peptides was replaced with a fresh medium. The cells werefurther cultured for 72 hours. The cells were analyzed for cellviability using WST-8. The results are shown in FIG. 24A.

Furthermore, after addition of TfR-lytic chimeric peptide atconcentration of 10 μM, the cells in calcein solution were observedafter 0 minute, five minutes, 10 minutes and 15 minutes. In T47D breastcancer cells, TfR-lytic chimeric peptide which permeated the membranewas observed (FIG. 24B).

Moreover, before the treatment with peptide (5 μM), T47D cells wereincubated with increasing concentration of anti-TfR monoclonal antibodyor nonspecific mouse IgG1 (isotype control) for three hours, whichdemonstrated inhibition of cell viability by TfR-lytic chimeric peptide(FIG. 25A).

(Cytotoxicity of siRNA or Scramble Sequence (sc) RNA to Cancer CellLines)

T47D and MDA-MB-231 cells were transfected with siRNA or scRNA, and fourdays after the transfection, the level of target gene in the cells wasanalyzed by flow cytometry analysis (data not shown). The inhibitionratio was assessed using WST-8 reagent. The assay was repeated threetimes, and the results are represented as mean of triplicatemeasurements±SD (bar). The results are shown in FIG. 25B.

(Possibility of TfR-Lytic Chimeric Peptide to Induce Cell Death Via anApoptotic Mechanism in Cancer Cells)

T47D and PE cells were incubated with TfR-lytic chimeric peptide (10 μM)and lytic peptide (10 μM) for two hours. Annexin V (FIG. 26A) andcaspase 3,7 activity (FIG. 26B) were detected in the green channel, andpropidium iodide staining was detected in the red channel, and flowcytometry analysis was performed. In T47D cells treated with TfR-lyticchimeric peptide, the ratio of the right half panel showing AnnexinV-positive cells and caspase 3,7-activated cells increased. On the otherhand, in normal cells PE, even with treatment with TfR-lytic chimericpeptide, the percentage of Annexin V-positive cells and caspase3,7-activated cells did not increase. It was suggested that TfR-lyticchimeric peptide induces cancer cell death by an apoptotic mechanism ina cancer cell-selective manner.

(Comparison of Action of Various Lytic Peptides to Induction of CellDeath by an Apoptotic Mechanism in Cancer Cell Lines)

T47D cells labeled with mitochondrial transmembrane potential-dependentfluorochrome JC-1 were untreated (untreated: upper left panel) ortreated with Staurosporine (control mitochondrial membrane potential:upper right panel), lytic peptide (lower left panel) or TfR-lyticchimeric peptide (lower right panel). After two hours, by flowcytometry, distinctive ratio in changes in red fluorescence or greenfluorescence indicating transmembrane potential was analyzed. Theresults are shown in FIG. 26C.

Furthermore, T47D cells were incubated with Staurosporine, TfR-lyticchimeric peptide (10 μM) and lytic peptide (10 μM). After two hours,cytoplasm extract was isolated, and by Western blot analysis using anantibody to cytochrome c, release of cytochrome c was investigated. Theresults of the Western blot analysis are shown in FIG. 26D.

(In Vivo Antitumor Activity of TfR-Lytic Chimeric Peptide)

MDA-MB-231 breast cancer cells were subcutaneously transplanted toathymic nude mice. As shown by arrow, from day 5, saline (control) orTfR-lytic peptide (0.3 mg/kg, 1 mg/kg or 3 mg/kg) was intratumorallyinjected. Each group was formed by three animals (n=3). The results ofmeasurement of tumor diameters over time are shown in FIG. 27A.

Furthermore, to the athymic nude mice which were subcutaneouslytransplanted with MDA-MB-231 breast cancer cells in the same manner, asshown by arrow, from day 5, saline (control) or TfR-lytic peptide (0mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg or 5 mg/kg) was intravenouslyinjected. Each group was formed by three animals (n=3). The results ofmeasurement of tumor diameters over time are shown in FIG. 27B.

In vivo antitumor activity of TfR-lytic chimeric peptide was observed.

Example 17 IL4-Lytic Chimeric Peptide

In the present Example, activity of IL4-Lytic chimeric peptide wasinvestigated.

(IL4-Lytic (L,D) Exhibits Selective Toxicity to Cancer Cells)

Using IL4-Lytic L formed only of L amino acids(KQLIRFLKRLDRNGGGKLLLKLLKKLLKLLKKK; SEQ ID NO: 18) and D,L-mixedIL4-Lytic (D,L) (KQLIRFLKRLDRNGGGKLLLKLLKKLLKLLKKK; SEQ ID NO: 44;underlined letters represent D-amino acids), cytotoxic activity tocancer cells was studied.

A total of 3×10³ cells per well of normal cell line WI-38 (A, B) andcancer cell line KCCT873 (C,D) were seeded in 96-well plates, culturedin a medium containing 10% FBS for 24 hours, and incubated withincreasing concentrations (0 to 30 μM) of IL4-lytic (L) chimericpeptide, lytic (L) peptide, IL-4-lytic (L,D) chimeric peptide or lytic(L,D) peptide in 1001 for 72 hours at 37° C. Cell viability was measuredwith WST-8 solution (Cell Count Reagent SF; Nakalai Tesque). The resultsare shown in FIG. 29 and Table 4.

TABLE 4 Cytotoxicity of peptides to various cell lines. IC₅₀ (μM) lyticIL4-lytic lytic IL4-lytic (L) (L) (L, D) (L, D) Cell lines Mean ± SDMean ± SD Mean ± SD Mean ± SD Normal cells PE 20> 20> 20> 20> HEK293T11.3 ± 1.1  7.2 ± 0.7  58 ± 0.3 14.2 ± 1.3  WI-38 14.4 ± 1.3  9.8 ± 0.8 100 ± 3.1 16.1 ± 0.5  Cancer cells BxPC-3 6.7 ± 0.4 3.2 ± 0.4 37.1 ±0.7 6.8 ± 0.3 MDA-MB-231 8.1 ± 0.4 5.6 ± 0.5 27.1 ± 1.5 5.7 ± 0.4 A1727.4 ± 0.3 3.8 ± 1.1 30.5 ± 0.9 6.8 ± 0.4 KCCT873 7.6 ± 0.8 4.6 ± 0.127.7 ± 1.4 5.9 ± 0.6 U251 6.5 ± 1.1 3.7 ± 0.1 17.2 ± 2.5 6.6 ± 0.4

From these results, it is recognized that use of D,L-mixed sequence wastherapeutically preferred in comparison with use of a sequenceconsisting only of L-amino acids. It was also confirmed that selectivitybetween cancer cells and normal cells was also retained.

(Detection of IL-4Rα Expression on Cell Surface of Cancer Cell Lines)

Total RNA from A172, BxPC-3 and normal cell line HEK293 werereverse-transcribed to cDNA, and then detection was performed byquantitative PCR using IL-4Rα-specific primer.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internalstandard. The results are shown in FIG. 30.

(IL4-Lytic (L,D) Chimeric peptide Rapidly Kills Cancer Cells)

PE cells (A), KCCT873 cells and BxPC-3 cells (B) were treated withIL4-lytic (L,D) chimeric peptide (black columns) or lytic (L,D) peptide(white columns) for two minutes, five minutes, 10 minutes, 30 minutes,one hour or two hours. The medium containing peptides was replaced witha fresh medium, and the cells were further cultured for 72 hours. Cellviability was determined using WST-8 reagent. The results are shown inFIG. 31.

(Possibility that Cancer Cell Death by IL4-Lytic (L,D) Chimeric PeptideMediates an Apoptotic Mechanism)

PE cells which are normal cells and KCCT873 cells which are cancer cellswere incubated with IL4-lytic (L,D) chimeric peptide or lytic (L,D)peptide (10 μM) for two hours. Subsequently, dual-color flow cytometryanalysis was performed for Annexin V labeling in green channel and PIstaining in red channel. The results are shown in FIG. 32. The valuesindicate percentage of cells in each quadrant is indicated.

(In Vivo Antitumor Activity of IR4-Lytic (L,D) Chimeric Peptide)

MDA-MB-231 breast cancer cells were subcutaneously transplanted toathymic nude mice. As shown by arrow, from day 5, saline (control) orIL4-lytic (L,D) peptide (0.5 mg/kg or 2 mg/kg) was intratumorallyinjected. Each group was formed by three animals (n=3). The results ofmeasurement of tumor diameters over time are shown in FIG. 33A.

Furthermore, to the athymic nude mice which were subcutaneouslytransplanted with MDA-MB-231 breast cancer cells in the same manner, asshown by arrow, from day 5, saline (control) or IL4-lytic (L,D) peptide(2 mg/kg or 5 mg/kg) was intravenously injected. Each group was formedby three animals (n=3). The results of measurement of tumor diametersover time are shown in FIG. 33B.

Example 18 Sema3A-nLytic Chimeric Peptide

In the present Example, activity of Sema3A-nLytic chimeric peptide wasinvestigated.

(Cytotoxic Activity of Sema3A-nLytic to Various Cell Lines)

Using newly designed nLytic peptide (LLKLLKKLLKKLLKL; SEQ ID NO: 45;underlined letters represent D-amino acids), Sema3A(aa363-377)-nLyticpeptide (NYQWVPYQGRVPYPRGGLLKLLKKLLKKLLKL; SEQ ID NO: 46; underlinedletters represent D-amino acids) and Sema3A(aa371-377)-nLytic peptide(D,L) (GRVPYPRGGLLKLLKKLLKKLLKL; SEQ ID NO: 47; underlined lettersrepresent D-amino acids), cytotoxic activity to cancer cells wasstudied.

A total of 3×10³ cells per well of pancreas cell line were seeded in96-well plates, cultured for 24 hours in a medium containing 10% FBS,and incubated with increasing concentrations (0 to 50 μM) ofSema3A-nLytic peptide or nLytic peptide alone in 100 μl for 48 hours at37° C. Cell viability was measured with WST-8 solution (Cell CountReagent SF; Nakalai Tesque). The results are shown in FIG. 34A and Table5.

TABLE 5 Cytotoxic activity of Sema3A-nLytic on pancreatic cell lines.IC₅₀ (μM) nLytic Sema3A(aa363-377)- Sema3A(aa371-377)- Cell lines alonenLytic nLytic Cancer cells BxPC-3 38.5* 12.5 14.7 CFPAC-1 22.6 10.6 9.2Panc-1 28.2 13.1 14.1 SU8686 19.5 7.9 8.0 Normal cellsPancreatic >50 >50 >50 epithelium *IC₅₀, the concentration of peptide atwhich 50% inhibition of cell viability is observed compared to untreatedcells.(Cytotoxic Activity of Sema3A-nLytic to Breast Cancer Cell Lines)

Breast cancer cells were cultured with various concentrations (0 to 20μM) of Sema3A (371-377)-nLytic or Sema3A (363-377)-nLytic for 48 hours,and cytotoxic activity was assessed with WST-8 reagent. The results areshown in FIG. 34B and Table 6.

TABLE 6 Cytotoxic activity of Sema3A-nLytic on breast cancer cell lines.IC₅₀ (μM) nLytic Sema3A(aa363-377)- Sema3A(aa371-377)- Cell lines alonenLytic nLytic BT20 38.0 10.9 11.4 MDA-MB-231 29.6 11.5 8.1 SKBR-3 8.014.5 T47D 12.5 4.1 3.7 *IC₅₀, the concentration of peptide at which 50%inhibition of cell viability is observed compared to untreated cells.

In normal cells, as shown in Table 5, IC₅₀ is a numerical value of >50,and the effect was attained at lower concentrations in the cancer cellsshown in Table 6. In view of this, it is believed that there isselectivity between normal cells and cancer cells.

(Cytotoxic Activity of Sema3A-nLytic to Various Cell Lines)

Various cells were cultured with various concentrations (0 to 20 μM) ofSema3A-nLytic (363-377) or Sema3A-nLytic (371-377) for 48 hours, andcytotoxic activity was assessed with WST-8 reagent. The assay wasrepeated three times, and the results are represented as mean oftriplicate measurements±SD (bar). The results are shown in FIG. 34C andTable 7.

TABLE 7 Cytotoxic activity of Sema3A-nLytic on various cell lines. IC₅₀(μM) Sema3A(aa363-377)- Sema3A(aa371-377)- Cell lines nLytic nLyticCancer cells HuCCT-1 13.6 7.9 Normal cells HEK293T >20 >20 *IC₅₀, theconcentration of peptide at which 50% inhibition of cell viability isobserved compared to untreated cells.

Furthermore, using BIACORE, wild-type peptide and some mutated peptidesof Sema3A were analyzed for interaction with neuropilin-1 (NRP-1). Theresults are shown in FIG. 35. (A) Binding of wild-type Sema3A peptide toNRP-1 protein. Serially diluted various concentrations (1.6 μM, 13 μM,26 μM) of wild-type Sema3A peptide samples were analyzed on parallelsensor surface. (B) Binding of wild-type peptide and mutated peptides ofSema3A (R372K and R372K/R377K) to NRP-1 protein. Various seriallydiluted wild-type Sema3A peptide samples (26 μM) were analyzed onparallel sensor surface. (C) Outline of binding ability of variousSema3A peptides to NRP-1 protein. (D) Peptide sequence of wild-typepeptide and mutated peptides of Sema3A.

(Expression of Neuropilin-1 in Pancreatic Cancer Cell Line and BreastCancer Cell Line)

Expression of neuropilin in some pancreatic cancer cell lines (BxPC-3,CFPAC-1, Panc-1 and SU8686) and pancreatic epithelial cells and breastcancer cells (BT-20, MDA-MB-231, SKBR-3 and T47D) was estimated byRT-PCR analysis. In all RT-PCR analyses, β-actin was used as positivecontrol. The results are shown in FIG. 36.

(Possibility that Cancer Cell Death by Sema3A-Lytic Mediates anApoptotic Mechanism)

PE cells (upper panel) and BxPC-3 cells (lower panel) were incubatedwith Sema3A-Lytic peptide (5 μM) for three hours at 37° C., and aftersix hours analyzed for Annexin V labeling by dual-color flow cytometry.The results are shown in FIG. 37. As apparent from the figure, in BxPC-3cells treated with Sema3A-nLytic, the ratio of the right half the panelwhich shows Annexin V-positive cells increased. On the other hand, innormal cells PE, the percentage of Annexin V-positive dead cells did notincrease even with treatment with Sema3A-nLytic. It was suggested thatSema3A-nLytic induces cancer cell death by an apoptotic mechanism in acancer cell-selective manner.

Example 19 Sema3A-kLytic Chimeric Peptide

In the present Example, using newly designed kLytic peptide(KLLLKLLKKLLKLLKKK; underlined letters represent D-amino acids; SEQ IDNO: 49) and Sema3A(aa363-377)-kLytic peptide(NYQWVPYQGRVPYPRGGGKLLLKLLKKLLKLLKKK; underlined letters representD-amino acids; SEQ ID NO 50), cytotoxic activity to cancer cells wasstudied.

A total of 3×10³ cells per well of pancreas cell line were seeded in96-well plates, cultured for 24 hours in a medium containing 10% FBS,and incubated with increasing concentrations (0 to 50 μM) ofSema3A-kLytic (aa366-377) peptide or kLytic peptide alone in 100 μl for48 hours at 37° C. Cell viability was measured with WST-8 solution (CellCount Reagent SF; Nakalai Tesque). The results are shown in FIG. 38 andTable 8.

TABLE 8 Cytotoxic activity of Sema3A-kLytic on pancreatic cancer cellsand normal cells. IC₅₀ (μM) Cell lines Sema3A(aa363-377)-kLytic kLyticCancer cells BxPC-3 5.7 ± 2.3 32 ± 1.6 CFPAC-1 6.7 ± 0.1 67 ± 0.9 Panc-1 13 ± 2.7 100< SU8686 5.2 ± 0.4 28 ± 0.5 Normal cells Pancreatic 20<epidermal cell MRC-5 50< 100< Human normal  37.5  50< hepatocyte cell*IC₅₀, the concentration of peptide at which 50% inhibition of cellviability is observed compared to untreated cells.

In view that the effect was attained at lower concentrations in cancercells as shown in Table 8, it is believed that there is selectivitybetween normal cells and cancer cells.

(Expression of Neurolilin-1 in Pancreatic Cancer Cell Lines)

Expression of neuropilin-1 in some pancreatic cancer cell lines (BxPC-3,Panc-1, SU8686 and CFPAC-1) and pancreatic epithelial cells wasestimated by RT-PCR analysis. In all RT-PCR analyses, GAPDH was used aspositive control. The results are shown in FIG. 39A.

Furthermore, for each cell, using real-time PCR, expression ofneuropilin-1 was determined. For all controls, analysis was performedusing GAPDH. The results are shown in FIG. 39B.

As apparent from FIGS. 39A and 39B, in SU8686 and CFPAC-1, expressionlevel of NRP-1 was high. In these cell lines, cell-killing effect ofSema3A (366-377)-kLytic peptide was excellent, and thus anticancereffect by a hybrid peptide targeted for NRP-1 can be expected.

(Possibility that Cancer Cell Death by Sema3A (363-377)-kLytic Mediatesan Apoptotic Mechanism)

It was studied whether or not Sema3A (363-377)-kLytic peptide inducesAnnexin V-positive expression in human pancreatic cancer cell lineSU8686 which highly expresses NRP-1.

SU8686 cells were incubated with Sema3A (363-377)-kLytic peptide (10 μM)for three hours at 37° C., and were analyzed for Annexin V labeling bydual-color flow cytometry. The results are shown in FIG. 40. As apparentfrom the figure, in SU8686 cells treated with Sema3A (363-377)-kLytic,the ratio of the right half panel which shows Annexin V-positive deadcells increased. It was suggested that Sema3A (363-377)-kLytic alsoinduces cell death of cancer cells by an apoptotic mechanism.

Specifically, from these results, anticancer tumor equivalent to that ofEB(H2R)-Lytic can be also expected for Sema3A(363-377)-kLytic peptide invivo.

(In Vivo Anticancer Action of (Sema3A(363-377)-kLytic Peptide)

(Protocol)

Human pancreatic cancer cell line BxPC-3 (5×10⁶ cells/150 μl phosphatebuffer) is subcutaneously injected to female 5-week-old nude micebalb/c-nu/nu. From day 5 after the transplantation,Sema3A(363-377)-kLytic peptide is intravenously administered three timesper week for three weeks, at 0, 0.5, 1, 2 or 5 mg/kg/50 μl phosphatebuffer per mouse. The tumor diameters are measured with electroniccaliper over time and the tumor volume (mm³) is calculated as longerdiameter×shorter diameter×shorter diameter×0.5.

Example 20 VEGFR2-Lytic Peptide

Using VEGFR2-lytic peptide ATWLPPRGGGKLLLKLLKKLLKLLKKK (underlinedletters represent D-amino acids; SEQ ID NO: 51), cytotoxic activity tocancer cells was studied.

A total of 3×10³ cells per well of five cancer cell lines (OE19, T47D,BxPC-3, U937 and LNCaP) and two normal cell lines (HEK293 and MRC-5)were seeded in 96-well plates, cultured for 24 hours in a mediumcontaining 10% FBS, and cultured with increasing concentration (0 to 20μM) of VEGFR2-lytic peptide in 100 μl for 72 hours. Cytotoxic activitywas assessed with WST-8 reagent (Cell Count Reagent SF; Nakalai Tesque).The results are shown in Table 9 and FIG. 41.

TABLE 9 Cytotoxic activity of VEGFR2-Lytic on various cancer cells andnormal cells. IC₅₀ (μM) Cell lines VEGFR2-Lytic Cancer cells OE19 7.1T47D 6.4 BxPC-3 12.2 U937 3.5 LNCaP 8.9 Normal cells HEK293 >20MRC-5 >20 *IC₅₀, the concentration of peptide at which 50% inhibition ofcell viability is observed compared to untreated cells.

As shown in the table, the effect was attained at lower concentration incancer cells. In view of this, it is believed that there is selectivitybetween normal cells and cancer cells.

(In Vivo Anticancer Action of VEGFR2-Lytic Peptide)

(Protocol)

Human pancreatic cancer cell line BxPC-3 (5×10⁶ cells/150 μl phosphatebuffer) is subcutaneously injected to female 5-week-old nude micebalb/c-nu/nu. From day 5 after the transplantation, VEGFR2-Lytic peptideis intravenously administered three times per week for three weeks, at0, 0.5, 1, 2 or 5 mg/kg/50 μl phosphate buffer per mouse. The tumordiameters are measured with electronic caliper over time and the tumorvolume (mm³) is calculated as longer diameter×shorter diameter×shorterdiameter×0.5.

Example 21 Test for Stability of Peptide

Stability of the chimeric peptides produced in Example 1 is studied.

(Protocol)

The peptidetoxins are stored for a short or long period at −20° C., 4°C. and 25° C. in saline or serum. Based on quantitative analysis by HPLCand cell-killing effect to cultured cancer cells and the like, chemicaland biological activities are assessed.

Example 22 A Method of Screening a Pharmaceutical/Anticancer Agent Usingan Amino Acid Sequence Targeting Both EGFR in Cancer Cells with HighEGFR Expression and the Cancer Cell Membrane of the Cancer Cells

In the present Example, a method of screening apharmaceutical/anticancer agent using an amino acid sequence targetingboth EGFR in cancer cells with high EGFR expression and the cancer cellmembrane is demonstrated.

(Protocol)

Cell membrane samples of cancer cells with high EGFR expression (humanlung cancer cell line H322 or the like) are prepared, and BIACOREanalysis is performed as follows. A test drug to be screened needs to bebiotinated.

Surface plasmon resonance (SPR) experiments are performed with a BIACOREbiosensor system 3000 (BIACORE Inc., Uppsala, Sweden). About 5000 RU ofstreptavidin (Sigma) is immobilized on the surface of CM5 sensor chipsvia N-hydroxysuccinimide andN-ethyl-N′-(dimethylaminopropyl)carbodiimide activation chemistry, andthen 2000-3000 RU of biotin-conjugated test drug, such as peptide, areinjected over the streptavidin-immobilized sensor chip. As a control ofnonspecific binding, the unreacted carboxymethyl groups of a sensor chipwithout immobilized streptavidin are blocked with ethanolamine. As ananalyte, cell surface proteins which are prepared using the Mem-PEReukaryotic membrane protein extraction reagent kit (Pierce) are injectedover the flow-cell in the flow rate of 20 μl/min. at 25° C. In order toprevent nonspecific binding during the assay, HBS buffer (0.01 M HEPES,0.15 M NaCl, 0.005% Tween 20, 3 mM EDTA [pH 7.4]) is used as runningbuffer. All protein concentrations used in these experiments aredetermined by the Bradford method (Bradford M M. A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizingthe principle of protein-dye binding. Anal Biochem 1976; 72:248-54).Data analysis is performed using BIA evaluation ver.3.2 software(BIACORE).

(Results)

The membrane samples of cancer cells with high EGFR expression arereacted with the sensor chip immobilized with the test drug.

In the case of test drug which bind to both EGFR and cancer cellmembrane, higher response is exhibited, and the test drug can bescreened.

As described above, the present invention has been illustrated by way ofpreferred embodiments of the invention, but the invention should not beinterpreted to be limited to these embodiments. It is understood thatthe scope of the present invention should be interpreted only based onthe claims. It is understood that those skilled in the art can carry outthe equivalent scope from the specific preferred embodiments based onthe description of the invention and common general knowledge. It isunderstood that the contents of the patent, patent applications anddocuments quoted herein should be incorporated herein as a reference forthe specification so that the contents per se are equivalent to bespecifically described herein.

INDUSTRIAL APPLICABILITY

The present invention provides an anticancer agent with side effectsalleviated.

[Sequence Listing Free Text]

SEQ ID NO: 1: amino acid sequence of a cancer cell membrane-lyticpeptide

KLLLKLLKKLLKLLKKK (underlined letters represent D-amino acids)

SEQ ID NO: 2: amino acid sequence of EB(EGFR-binding)-cancer cellmembrane-lytic chimeric peptide

YHWYGYTPQNVIGGGKLLLKLLKKLLKLLKKK (underlined letters represent D-aminoacids)

SEQ ID NO: 3: amino acid sequence of recombinant human EGF receptor(rhEGFR) NP_(—)005219 (among 1210AA, remaining 1186AA except for theunderlined. N-terminal 24AA)

SEQ ID NO: 4: polycathionic amino acid sequence of mitochondriotoxicpeptide and apoptosis-inducing peptide KLAKLAKKLAKLAK

SEQ ID NO: 5: amino acid sequence of fibroblast growth factor receptor(FGFR)-binding peptide: MQLPLAT

SEQ ID NO: 6: amino acid sequence of fibroblast growth factor receptor(FGFR)-binding peptide: AAVALLPAVLLALLAP

SEQ ID NO: 7: amino acid sequence which is EGF receptor-binding peptide:YHWYGYTPQNVI

SEQ ID NO: 8: amino acid sequence which is EGF receptor-binding peptide:X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂

SEQ ID NO: 9: YRWYGYTPQNVI

SEQ ID NO: 10: YKWYGYTPQNVI

SEQ ID NO: 11: FLKLLKKLAAKLF

SEQ ID NO: 12: RLLRRLLRRLLRRLLRRLLR

SEQ ID NO: 13: RLLRRLLRRLLRK

SEQ ID NO: 14: EB(H2R)-Lytic: YRWYGYTPQNVIGGGKLLLKLLKKLLKLLKKK

SEQ ID NO: 15:HER2-Lytic:YCDGFYACYMDVGGGKLLLKLLKKLLKLLKKK (underlinedletters represent D-amino acids and the remaining are L-amino acids)

SEQ ID NO: 16: VEGFR1-Lytic: WHSDMEWWYLLGGGGKLLLKLLKKLLKLLKKK(underlined letters represent D-amino acids and the remaining areL-amino acids)

SEQ ID NO: 17: TfR-Lytic: THRPPMWSPVWPGGGKLLLKLLKKLLKLLKKK (underlinedletters represent D-amino acids and the remaining are L-amino acids)

SEQ ID NO: 18: IL4-LyticL: KQLIRFLKRLDRNGGGKLLLKLLKKLLKLLKKK

SEQ ID NO: 19: IL13-LyticL: KDLLLHLKKLFREGQFNGGGKLLLKLLKKLLKLKKK

SEQ ID NO: 20: Sema3A-LyticL<binding to human neuropilin-1>:NYQWVPYQGRVPYPRGGGKLLLKLLKKLLKLLKKK

SEQ ID NO: 21: EGFbuf: YHWYGYTPQNVIGGGGGRLLRRLLRRLLRK

SEQ ID NO: 22: amino acid sequence of interleukin-11 receptor(IL11R)-binding peptide:CGRRAGGSC (cyclic)

SEQ ID NO: 23: cell-permeable peptide TA sequence: YGRKKRRQRRR

SEQ ID NO: 24: cell-permeable peptide R11 sequence: RRRRRRRRRRR

SEQ ID NO: 25: cytotoxic peptide: RQIKIQFQNRRMKWKKKAYARIGNSYFK

SEQ ID NO: 26: IL4R-binding peptide sequence: KQLIRFLKRLDRN

SEQ ID NO: 27: LyticL: KLLLKLLKKLLKLLKKK (L-amino acids alone)

SEQ ID NO: 28: IL13R-binding peptide sequence: KDLLLHLKKLFREGQFN

SEQ ID NO: 29: sequence necessary for binding to neuropilinreceptor-binding peptide (sequence important for binding to ligandSema3A):NYQWVPYQGRVPYPR

SEQ ID NO: 30: human epidermal growth factor receptor 2 (HER2)-bindingpeptide sequence:YCDGFYACYMDV

SEQ ID NO: 31: amino acid sequence of vascular epithelial growth factorreceptor 1 (VEGFR1)-binding peptide: WHSDMEWWYLLG

SEQ ID NO: 32: amino acid sequence of vascular epithelial growth factorreceptor 1 (VEGFR1)-binding peptide: VEPNCDIHVMWEWECFERL

SEQ ID NO: 33: amino acid sequence of vascular epithelial growth factorreceptor (VEGFR)-binding peptide: GGNECDAIRMWEWECFERL

SEQ ID NO: 34: amino acid sequence of transferrin receptor (TfR)-bindingpeptide:THRPPMWSPVWP

SEQ ID NO: 35: amino acid sequence of prostate-specific membrane antigen(PSMA)-binding peptide: CQKHHNYLC

SEQ ID NO: 36: amino acid sequence of neuropilin-1 (NRP1)/vascularendothelial growth factor receptor 2 (VEGFR2) binding peptide: ATWLPPR

SEQ ID NO: 37: amino acid sequence of ephrin B1 (EphB1)-binding peptide:EWLS

SEQ ID NO: 38: amino acid sequence ephrin B2 (EphB2) binding peptide:SNEW

SEQ ID NO: 39: amino acid sequence of binding peptide of glucoseregulation protein 78 (GRP78): WDLAWMFRLPVG

SEQ ID NO: 40: amino acid sequence of glucose regulation protein 78(GRP78) binding peptide: CTVALPGGYVRVC (cyclic)

SEQ ID NO: 41: original Lytic peptide: LKLLKKLLKKLLKLL-NH₂ (underlinedletters represent D-amino acids and the remaining are L-amino acids)

SEQ ID NO: 42: EB-original Lytic:YHWYGYTPQNVIGGG LKLLKKLLKKLLKLL-NH₂(underlined letters represent D-amino acids and the remaining areL-amino acids)

SEQ ID NO: 43: EB(H2R)-Lytic: YRWYGYTPQNVIGGG KLLLKLLKKLLKLLKKK(underlined letters represent D-amino acids and the remaining areL-amino acids)

SEQ ID NO: 44: IL4-Lytic(D,L): KQLIRFLKRLDRNGGG KLLLKLLKKLLKLLKKK(underlined letters represent D-amino acids and the remaining areL-amino acids)

SEQ ID NO: 45:nLytic:LLKLLKKLLKKLLKL (underlined letters representD-amino acids and the remaining are L-amino acids)

SEQ ID NO: 46: Sema3A(aa363-377)-nLytic:NYQWVPYQGRVPYPRGGLLKLLKKLLKKLLKL (underlined letters represent D-aminoacids and the remaining are L-amino acids)

SEQ ID NO: 47: Sema3A(aa371-377)-nLytic: GRVPYPRGGLLKLLKKLLKKLLKL(underlined letters represent D-amino acids and the remaining areL-amino acids)

SEQ ID NO: 48: KLLLKLLKKLLKLLKKK (wherein each amino acid isindependently L-, D-, or D,L,-mixed amino acid)

SEQ ID NO: 49: kLytic peptide:KLLLKLLKKLLKLLKKK (underlined lettersrepresent D-amino acids and the remaining are L-amino acids)

SEQ ID NO: 50: Sema3A(363-377)-kLytic peptide:NYQWVPYQGRVPYPRGGGKLLLKLLKKLLKLLKKK (underlined letters representD-amino acids and the remaining are L-amino acids)

SEQ ID NO: 51:VEGFR2-lytic peptide: ATWLPPRGGGKLLLKLLKKLLKLLKKK(underlined letters represent D-amino acids and the remaining areL-amino acids)

SEQ ID NO: 52: human epidermal growth factor receptor type 2(HER2)-binding peptide sequence: LLGPYELWELSH

SEQ ID NO: 53: human epidermal growth factor receptor type 2(HER2)-binding peptide sequence: ALVRYKDPLFVWGFL

SEQ ID NO: 54: human epidermal growth factor receptor type 2(HER2)-binding peptide sequence: KCCYSL

SEQ ID NO: 55: human epidermal growth factor receptor type 2(HER2)-binding peptide sequence: WTGWCLNPEESTWGFCTGSF

SEQ ID NO: 56: human epidermal growth factor receptor type 2(HER2)-binding peptide sequence: DTDMCWWWSREFGWECAGAG

SEQ ID NO: 57: amino acid sequence of Tat [human immunodeficiency virus1](NP_(—)057853): MEPVDPRLEP WKHPGSQPKT ACTNCYCKKC CFHCQVCFIT KALGISYGRKKRRQRRRAHQ NSQTHQASLS KQPTSQPRGD PTGPKE

The invention claimed is:
 1. A chimeric peptide comprising areceptor-binding peptide and a cytotoxic peptide, wherein thereceptor-binding peptide is a neuropilin receptor-binding peptidecomprising the amino acid sequence set forth in any one of SEQ ID NOS:29 and 60-67, and wherein the cytotoxic peptide is cell membrane-lyticpeptide comprising a 10- to 20-amino acid sequence consisting only of Kand L, and the amino acids are L-, D- or a mixture of D- and L-aminoacids.
 2. The chimeric peptide according to claim 1, wherein the cellmembrane-lytic peptide is the amino acid sequence set forth in SEQ IDNO: 48, and the amino acids are L-, D- or a mixture of D- and aminoacids.
 3. The chimeric peptide according to claim 1, wherein thecytotoxic peptide is the amino acid sequence set forth in SEQ ID NO: 1.4. The chimeric peptide according to claim 1, further comprising aspacer peptide.
 5. The chimeric peptide according to claim 4, whereinthe spacer peptide is a sequence of at least one amino acid in lengthand wherein the spacer peptide is a sequence in which up to 5 of glycineresidues, proline residues or a mixture thereof are linked.
 6. Thechimeric peptide according to claim 4, wherein the spacer peptide isGGG.
 7. The chimeric peptide according to claim 1 having the amino acidsequence set forth in SEQ ID NO:
 20. 8. A pharmaceutical compositioncomprising the chimeric peptide according to claim
 1. 9. An anticanceragent comprising the chimeric peptide according to claim
 1. 10. Anucleic acid encoding the chimeric peptide according to claim
 1. 11. Avector comprising a nucleic acid which encodes the chimeric peptideaccording to claim
 1. 12. An isolated host cell comprising a nucleicacid which encodes the chimeric peptide according to claim
 1. 13. Amethod of treating cancer which overexpresses neuropilin receptorcomprising the step of administering the chimeric peptide according toclaim 1.